IRNA agents with biocleavable tethers

ABSTRACT

The invention relates to iRNA agents, which preferably include a monomer in which the ribose moiety has been replaced by a moiety other than ribose that further includes a tether having one or more linking groups, in which at least one of the linking groups is a cleavable linking group. The tether in turn can be connected to a selected moiety, e.g., a ligand, e.g., a targeting or delivery moiety, or a moiety which alters a physical property. The cleavable linking group is one which is sufficiently stable outside the cell such that it allows targeting of a therapeutically beneficial amount of an iRNA agent (e.g., a single stranded or double stranded iRNA agent), coupled by way of the cleavable linking group to a targeting agent—to targets cells, but which upon entry into a target cell is cleaved to release the iRNA agent from the targeting agent.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. application Ser. No.10/916,185, filed Aug. 10, 2004, which is a continuation-in-part ofInternational Application No. PCT/US2004/011829, filed on Apr. 16, 2004,which claims the benefit of U.S. Provisional Application No. 60/493,986,filed on Aug. 8, 2003; U.S. Provisional Application No. 60/494,597,filed on Aug. 11, 2003; U.S. Provisional Application No. 60/506,341,filed on Sep. 26, 2003; U.S. Provisional Application No. 60/518,453,filed on Nov. 7, 2003; U.S. Provisional Application No. 60/463,772,filed on Apr. 17, 2003; U.S. Provisional Application No. 60/465,802,filed on Apr. 25, 2003; U.S. Provisional Application No. 60/469,612,filed on May 9, 2003; U.S. Provisional Application No. 60/510,246, filedon Oct. 9, 2003; U.S. Provisional Application No. 60/510,318, filed onOct. 10, 2003; U.S. Provisional Application No. 60/503,414, filed onSep. 15, 2003; U.S. Provisional Application No. 60/465,665, filed onApr. 25, 2003; International Application No. PCT/US04/07070, filed onMar. 8, 2004; International Application No. PCT/US2004/10586, filed onApr. 5, 2004; International Application No. PCT/US2004/11255, filed onApr. 9, 2004; and International Application No. PCT/US2004/011822, filedon Apr. 16, 2004. The contents of all of these prior applications arehereby incorporated by reference in their entireties.

TECHNICAL FIELD

The invention relates to iRNA agents, which preferably include a monomerin which the ribose moiety has been replaced by a moiety other thanribose. The monomer can be used to attach a ligand, e.g., a lipophilicmoiety, such as cholesterol, directly or indirectly to the iRNA agentvia a tether that includes a cleavable linking group. The invention alsorelates to methods of making and using such modified iRNA agents.

BACKGROUND

RNA interference or “RNAi” is a term initially coined by Fire andco-workers to describe the observation that double-stranded RNA (dsRNA)can block gene expression when it is introduced into worms (Fire et al.(1998) Nature 391, 806-811). Short dsRNA directs gene-specific,post-transcriptional silencing in many organisms, including vertebrates,and has provided a new tool for studying gene function. RNAi may involvemRNA degradation.

SUMMARY

The inventor has discovered, inter alia, that the ribose sugar of one ormore ribonucleotide subunits of an iRNA agent can be replaced withanother moiety, e.g., a non-carbohydrate (preferably cyclic) carrierthat further includes a tether having one or more linking groups, inwhich at least one of the linking groups is a cleavable linking group.The tether in turn can be connected to a selected moiety, e.g., aligand, e.g., a targeting or delivery moiety, or a moiety which alters aphysical property. The cleavable linking group is one which issufficiently stable outside the cell such that it allows targeting of atherapeutically beneficial amount of an iRNA agent (e.g., a singlestranded or double stranded iRNA agent), coupled by way of the cleavablelinking group to a targeting agent—to targets cells, but which uponentry into a target cell is cleaved to release the iRNA agent from thetargeting agent.

A ribonucleotide subunit in which the ribose sugar of the subunit hasbeen so replaced is referred to herein as a ribose replacementmodification subunit (RRMS). A cyclic carrier may be a carbocyclic ringsystem, i.e., all ring atoms are carbon atoms, or a heterocyclic ringsystem, i.e., one or more ring atoms may be a heteroatom, e.g.,nitrogen, oxygen, sulfur. The cyclic carrier may be a monocyclic ringsystem, or may contain two or more rings, e.g. fused rings. The cycliccarrier may be a fully saturated ring system, or it may contain one ormore double bonds.

The carriers further include (i) at least two “backbone attachmentpoints” and (ii) at least one “tethering attachment point.” A “backboneattachment point” as used herein refers to a functional group, e.g. ahydroxyl group, or generally, a bond available for, and that is suitablefor incorporation of the carrier into the backbone, e.g., the phosphate,or modified phosphate, e.g., sulfur containing, backbone, of aribonucleic acid. A “tethering attachment point” in some embodimentsrefers to a constituent ring atom of the cyclic carrier, e.g., a carbonatom or a heteroatom (distinct from an atom which provides a backboneattachment point), that connects the tethers described above or atethered moiety, e.g., a ligand, e.g., a targeting or delivery moiety,or a moiety which alters a physical property. One of the most preferredtethered moieties is a moiety which promotes entry into a cell, e.g., alipophilic moiety, e.g., cholesterol. While not wishing to be bound bytheory it is believed the attachment of a lipohilic agent increases thelipophilicity of an iRNA agent. Optionally, the selected moiety isconnected by an intervening tether to the cyclic carrier. Thus, it willoften include a functional group, e.g., an amino group, or generally,provide a bond, that is suitable for incorporation or tethering ofanother chemical entity, e.g., a ligand to the constituent ring.

Incorporation of one or more ligand conjugated monomer subunits(sometimes referred to herein as ribose replacement monomer subunits,RRMSs) described herein into an RNA agent, e.g., an iRNA agent,particularly when tethered to an appropriate entity, can confer one ormore new properties to the RNA agent and/or alter, enhance or modulateone or more existing properties in the RNA molecule. E.g., it can alterone or more of lipophilicity or nuclease resistance. Incorporation ofone or more RRMSs described herein into an iRNA agent can, particularlywhen the RRMS is tethered to an appropriate entity, modulate, e.g.,increase, binding affinity of an iRNA agent to a target mRNA, change thegeometry of the duplex form of the iRNA agent, alter distribution ortarget the iRNA agent to a particular part of the body, or modify theinteraction with nucleic acid binding proteins (e.g., during RISCformation and strand separation).

Accordingly, in one aspect, the invention features, an iRNA agentpreferably comprising a first strand and optionally a second strand,wherein at least one subunit having a formula (I) is incorporated intoat least one of said strands:

wherein:

X is N(CO)R⁷, NR⁷ or CH₂;

Y is NR⁸, O, S, CR⁹R¹⁰, or absent;

Z is CR¹¹R¹² or absent;

Each of R¹, R², R³, R⁴, R⁹, and R¹⁰ is, independently, H, OR^(a),OR^(b), (CH₂)_(n)OR^(a), or (CH₂)_(n)OR^(b), provided that at least oneof R¹, R², R³, R⁴, R⁹, and R¹⁰ is OR^(a) or OR^(b) and that at least oneof R¹, R², R³, R⁴, R⁹, and R¹⁰ is (CH₂)_(n)OR^(a), or (CH₂)_(n)OR^(b)(when the RRMS is terminal, one of R¹, R², R³, R⁴, R⁹, and R¹⁰ willinclude R^(a) and one will include R^(b); when the RRMSS is internal,two of R¹, R², R³, R⁴, R⁹, and R¹⁰ will each include an R^(b)); furtherprovided that preferably OR^(a) may only be present with (CH₂)_(n)OR^(b)and (CH₂)_(n)OR^(a) may only be present with OR^(b);

Each of R⁵, R⁶, R¹¹, and R¹² is, independently, H, C₁-C₆ alkyloptionally substituted with 1-3 R¹³, or C(O)NHR⁷; or R⁵ and R¹¹ togetherare C₃-C₈ cycloalkyl optionally substituted with R¹⁴;

R⁷ can be T-R^(d), in which T is a tether having one or more linkinggroups, wherein at least one of the linking groups is cleavable; andR^(d) can be H or a ligand e.g., a lipophilic ligand, e.g., cholesterol;

R⁸ is C₁-C₆ alkyl;

R¹³ is hydroxy, C₁-C₄ alkoxy, or halo;

R¹⁴ is NR^(c)R⁷;

Each of A and C is, independently, O or S;

B is OH, O⁻, or

R^(c) is H or C₁-C₆ alkyl; and

n is 1-4; provided that X is N(CO)R⁷ or NR⁷; or one of R⁵, R⁶, R¹¹, andR¹² is C(O)NHR⁷; or R¹⁴ is present.

Embodiments can include one or more of the following features.

The linking groups can be cleaved at least about 100 times (e.g., about90 times faster, about 80 times faster, about 70 times faster, about 60times faster, about 50 times faster, about 40 times faster, about 30times faster, about 20 times faster, about 10 times faster) faster inintracellular media (e.g., under conditions chosen to mimicintracellular media) than in extracellular media (e.g., under conditionschosen to mimic extracellular media).

In some embodiments, at least one of the linking groups can be a redoxcleavable linking group, an acid cleavable linking group, an esterasecleavable linking group, a phosphatase cleavable linking group, or apeptidase cleavable linking group.

In some embodiments, at least one of the linking groups can be areductively cleavable linking group (e.g., a disulfide group).

In some embodiments, at least one of the linking groups can be an acidcleavable linking group (e.g., a hydrazone group or an ester group).

In some embodiments, at least one of the linking groups can be anesterase cleavable linking group (e.g., an ester group).

In some embodiments, at least one of the linking groups can be aphosphatase cleavable linking group (e.g., a phosphate group).

In some embodiments, at least one of the linking groups can be anpeptidase cleavable linking group (e.g., a peptide bond).

T can include a terminal linking group (e.g., a terminal linking groupthat links the tether to the ligand or a terminal linking group thatlinks the tether to the nitrogen atom of X or R¹⁴ in formula (I), or thenitrogen atom of CONHR⁷ when R⁵, R⁶, R¹¹, or R¹² is CONHR⁷ in formula(I).

T can include a terminal linking group that links the tether to theligand and a terminal linking group that links the tether to thenitrogen atom of X or R¹⁴ in formula (I), or the nitrogen atom of CONHR⁷when R⁵, R⁶, R¹¹, or R¹² is CONHR⁷ in formula (I).

T can include one or more internal linking groups (e.g., 1-20 internallinking groups, 1-15 internal linking groups, 1-10 internal linkinggroups, 1-5 internal linking groups, 1-3 internal linking groups, 1-2linking groups).

T can include a terminal linking group (e.g., a terminal linking groupthat links the tether to the ligand and/or a terminal linking group thatlinks the tether to the nitrogen atom of X or R¹⁴ in formula (I), or thenitrogen atom of CONHR⁷ when R⁵, R⁶, R¹¹, or R¹² is CONHR⁷ in formula(I)) and one or more internal linking groups (e.g., 1-5, e.g., 1, 2, 3,4, or 5 internal linking groups).

The terminal linking group can be a cleavable linking group.

T can include at least one internal linking group that is cleavable.

T can include a cleavable terminal linking group that links the tetherto the ligand and/or a cleavable terminal linking group links the tetherto the nitrogen atom of X or R¹⁴ in formula (I), or the nitrogen atom ofCONHR⁷ when R⁵, R⁶, R¹¹, or R¹² is CONHR⁷ in formula (I), and/or atleast one internal linking group that is cleavable.

The terminal linking group that links the tether to the ligand can becleavable.

The terminal linking group that links the tether to the nitrogen atom ofX or R¹⁴ in formula (I), or the nitrogen atom of CONHR⁷ when R⁵, R⁶,R¹¹, or R¹² is CONHR⁷ in formula (I) can be cleavable.

T can include at least one internal linking group that is cleavable.

R⁷ can have the formula R^(d)-(E′)_(s)-Δ-(E″)_(t)-;

-   -   wherein:    -   each of E′ and E″ is a terminal linking group; and    -   Δ is a hydrocarbon chain that optionally includes one or more        internal linking groups, G;    -   each of s and t is, independently, 0 or 1;

provided that one of s or t is 1, or Δ includes at least one G;

s can be 1 and t can be 0.

s can be 0, and t can be 1.

s and t can both be 1.

Each of E′, E″, and G can be, independently,

—NR^(k)C(O)—, —C(O)NR^(k)—, —OC(O)NR^(k)—, —NR^(k)C(O)O—, —O—, —S—,—SS—, —S(O)—, —S(O₂)—, —NR^(k)C(O)NR^(k)—, —NR^(k)C(S)NR^(k)—, —C(O)O—,—OC(O)—, —NR^(k)C(S)—, —NR^(k)C(S)O—, —C(S)NR^(k)—, —OC(S)NR^(k)—,—NR^(k)C(S)O—, —O—P(O)(OR^(k))—O—, —O—P(S)(OR^(k))—O—,—O—P(S)(SR^(k))—O—, —S—P(O)(OR^(k))—O—, —O—P(O)(OR^(k))—S—,—S—P(O)(OR^(k))—S—, —O—P(S)(OR^(k))—S—, —S—P(S)(OR^(k))—O—,—O—P(O)(R^(k))—O—, —O—P(S)(R^(k))—O—, —S—P(O)(R^(k))—O—,—S—P(S)(R^(k))—O—, —S—P(O)(R^(k))—S—, —O—P(S)(R^(k))—S—, —C(O)—,—NR^(k)— —R^(k)C═NNR^(k)—, —NHCHR^(k′)C(O)NHCHR^(k″)C(O)—, or—NHCHR^(k′)NHC(O)CHR^(k″)C(O)—; in which

R^(k) at each occurrence can be, independently, C1-C20 alkyl, C1-C20haloalkyl, C6-C10 aryl, C7-C12 aralkyl; and

each of R^(k′) and R^(k″) can be, independently of one another, an aminoacid sidechain.

In certain embodiments, at least one of E′, E″, and G can be —SS—,—O—P(O)(OR^(k))—O—, —O—P(S)(OR^(k))—O—, —O—P(S)(SR^(k))—O—,—S—P(O)(OR^(k))—O—, —O—P(O)(OR^(k))—S—, —S—P(O)(OR^(k))—S—,—O—P(S)(OR^(k))—S—, —S—P(S)(OR^(k))—O—, —O—P(O)(R^(k))—O—,—O—P(S)(R^(k))—O—, —S—P(O)(R^(k))—O—, —S—P(S)(R^(k))—O—,—S—P(O)(R^(k))—S—, —O—P(S)(R^(k))—S—, —C(O)O—, —OC(O)—,—R^(k)C═NNR^(k)—, —NHCHR^(k′)C(O)NHCHR^(k″)C(O)—, or—NHCHR^(k′)NHC(O)CHR^(k″)C(O)—; and the others can each be,independently, NR^(k)C(O)—, —C(O)NR^(k)—, —OC(O)NR^(k)—, —NR^(k)C(O)O—,—O—, —S—, NR^(k)C(O)NR^(k)—, —NR^(k)C(S)NR^(k)—, —NR^(k)C(S)—,—NR^(k)C(S)O—, —C(S)NR^(k)—, —OC(S)NR^(k)—, —NR^(k)C(S)O, —C(O)—,—NR^(k)—.

In certain embodiments, at least one G can be —SS—, —O—P(O)(OR^(k))—O—,—O—P(S)(OR^(k))—O—, —O—P(S)(SR^(k))—O—, —S—P(O)(OR^(k))—O—,—O—P(O)(OR^(k))—S—, —S—P(O)(OR^(k))—S—, —O—P(S)(OR^(k))—S—,—S—P(S)(OR^(k))—O—, —O—P(O)(R^(k))—O—, —O—P(S)(R^(k))—O—,—S—P(O)(R^(k))—O—, —S—P(S)(R^(k))—O—, —S—P(O)(R^(k))—S—,—O—P(S)(R^(k))—S—, —C(O)O—, —OC(O)—, or —R^(k)C═NNR^(k)—,—NHCHR^(k′)C(O)NHCHR^(k″)C(O)—, or —NHCHR^(k′)NHC(O)CHR^(k″)C(O)—.

In certain embodiments, at least one G can be —SS—.

In certain embodiments, at least one G can be —C(O)O—, or —OC(O)—.

In certain embodiments, at least one G can be —R^(k)C═NNR^(k)—.

In certain embodiments, at least one G can be—NHCHR^(k′)C(O)NHCHR^(k″)C(O)—, or —NHCHR^(k′)NHC(O)CHR^(k″)C(O)—.

In certain embodiments, at least one G can be —O—P(O)(OR^(k))—O—,—O—P(S)(OR^(k))—O—, —O—P(S)(SR^(k))—O—, —S—P(O)(OR^(k))—O—,—O—P(O)(OR^(k))—S—, —S—P(O)(OR^(k))—S—, —O—P(S)(OR^(k))—S—,—S—P(S)(OR^(k))—O—, —O—P(O)(R^(k))—O—, —O—P(S)(R^(k))—O—,—S—P(O)(R^(k))—O—, —S—P(S)(R^(k))—O—, —S—P(O)(R^(k))—S—,—O—P(S)(R^(k))—S—.

Δ can be C1-100 alkylene, alkenylene, or alkynylene having at least oneG.

Δ can be C1-20 alkylene, alkenylene, or alkynylene having at least oneG.

Δ can be C1-20 alkylene, alkenylene, or alkynylene having 1, 2, 3, 4, or5 G.

Δ can be C1-20 alkylene having at least one G.

Δ can be C16 alkylene, C14 alkylene, or C12 alkylene having 1, 2, 3, or4 G.

Δ can be C9 alkylene chain having 1, 2, or 3 G.

Δ can be a C5 alkylene having 1 or 2 G.

Δ can be C18 alkylene chain having 1, 2, 3, 4, or 5 G.

-(E′)_(s)-Δ-(E″)_(t)- can be

The iRNA agent can be 21 nucleotides in length and there can be a duplexregion of about 19 pairs.

The iRNA agent can include a duplex region between 17 and 23 pairs inlength.

R¹ can be CH₂OR^(a) and R³ can be OR^(b); or R¹ can be CH₂OR^(a) and R⁹can be OR^(b) or R¹ can be CH₂OR^(a) and R² can be OR^(b).

R¹ can be CH₂OR^(b) and R³ can be OR^(b); or R¹ can be CH₂OR^(b) and R⁹can be OR^(b); or R¹ can be CH₂OR^(b) and R² can be OR^(b); or R¹ can beCH₂OR^(b) and R³ can be OR^(a); or R¹ can be CH₂OR^(b) and R⁹ can beOR^(a); or R¹ can be CH₂OR^(b) and R² can be OR^(a).

R¹ can be OR^(a) and R³ can be CH₂OR^(b); or R¹ can be OR^(a) and R⁹ canbe CH₂OR^(b); or R¹ can be OR^(a) and R² can be CH₂OR^(b).

R¹ can be OR^(b) and R³ can be CH₂OR^(b); or R¹ can be OR^(b) and R⁹ canbe CH₂OR^(b); or R¹ can be OR^(b) and R² can be CH₂OR^(b); or R¹ can beOR^(b) and R³ can be CH₂OR^(a); or R¹ can be OR^(b) and R⁹ can beCH₂OR^(a); or R¹ can be OR^(b) and R² can be CH₂OR^(a).

R³ can be CH₂OR^(a) and R⁹ can be OR^(b); or R³ can be CH₂OR^(a) and R⁴can be OR^(b).

R³ can be CH₂OR^(b) and R⁹ can be OR^(b); or R³ can be CH₂OR^(b) and R⁴can be OR^(b); or R³ can be CH₂OR^(b) and R⁹ can be OR^(a); or R³ can beCH₂OR^(b) and R⁴ can be OR^(a).

R³ can be OR^(b) and R⁹ can be CH₂OR^(a); or R³ can be OR^(b) and R⁴ canbe CH₂OR^(a); or R³ can be OR^(b) and R⁹ can be CH₂OR^(b); or R³ can beOR^(b) and R⁴ can be CH₂OR^(b).

R³ can be OR^(a) and R⁹ can be CH₂OR^(b); or R³ can be OR^(a) and R⁴ canbe CH₂OR^(b).

R⁹ can be CH₂OR^(a) and R¹⁰ can be OR^(b).

R⁹ can be CH₂OR^(b) and R¹⁰ can be OR^(b); or R⁹ can be CH₂OR^(b) andR¹⁰ can be OR^(a).

In a preferred embodiment the ribose is replaced with a pyrrolinescaffold or with a 4-hydroxyproline-derived scaffold, and X is N(CO)R⁷or NR⁷, Y is CR⁹R¹⁰, and Z is absent.

R¹ and R³ can be cis or R¹ and R³ can be trans.

n can be 1.

A can be O or S.

R¹ can be (CH₂)_(n)OR^(b) and R³ can be OR^(b); or R¹ can be(CH₂)_(n)OR^(a) and R³ can be OR^(b).

R⁷ can be can be T-R^(d), in which T is a tether having one or morelinking groups, wherein at least one of the linking groups is cleavable;and R^(d) can be H or a ligand, e.g., R^(d) can be chosen from a folicacid radical; a cholesterol radical; a carbohydrate radical; a vitamin Aradical; a vitamin E radical; a vitamin K radical. Preferably, R^(d) isa cholesterol radical.

R¹ can be OR^(b) and R³ can be (CH₂)_(n)OR^(b); or R¹ can be OR^(b) andR³ can be (CH₂)_(n)OR^(a); or R¹ can be OR^(a) and R³ can be(CH₂)_(n)OR^(b); or R¹ can be (CH₂)_(n)OR^(b) and R⁹ can be OR^(a).

R¹ and R⁹ can be cis or R¹ and R⁹ can be trans.

R¹ can be OR^(a) and R⁹ can be (CH₂)_(n)OR^(b); or R¹ can be(CH₂)_(n)OR^(b) and R⁹ can be OR^(b); or R¹ can be (CH₂)_(n)OR^(a) andR⁹ can be OR^(b); or R¹ can be OR^(b) and R⁹ can be (CH₂)_(n)OR^(b); orR¹ can be OR^(b) and R⁹ can be (CH₂)_(n)OR^(a).

R³ can be (CH₂)_(n)OR^(b) and R⁹ can be OR^(a); or R³ can be(CH₂)_(n)OR^(b) and R⁹ can be OR^(b); or R³ can be (CH₂)_(n)OR^(a) andR⁹ can be OR^(b); or R³ can be OR^(a) and R⁹ can be (CH₂)_(n)OR^(b); R³can be OR^(b) and R⁹ can be (CH₂)_(n)OR^(b); or R³ can be OR^(b) and R⁹can be (CH₂)_(n)OR^(a).

R³ and R⁹ can be cis or R³ and R⁹ can be trans.

In other preferred embodiments the ribose is replaced with a piperidinescaffold, and X is N(CO)R⁷ or NR⁷, Y is CR⁹R¹⁰, and Z is CR¹¹R¹².

R⁹ can be (CH₂)_(n)OR^(b) and R¹⁰ can be OR^(a).

n can be 1 or 2.

R⁹ can be (CH₂)_(n)OR^(b) and R¹⁰ can be OR^(b); or R⁹ can be(CH₂)_(n)OR^(a) and R¹⁰ can be OR^(b).

A can be O or S.

R⁷ can be can be T-R^(d), in which T is a tether having one or morelinking groups, wherein at least one of the linking groups is cleavable;and R^(d) can be H or a ligand, e.g., R^(d) can be chosen from a folicacid radical; a cholesterol radical; a carbohydrate radical; a vitamin Aradical; a vitamin E radical; a vitamin K radical. Preferably, R^(d) isa cholesterol radical.

R³ can be (CH₂)_(n)OR^(b) and R⁴ can be OR^(a); or R³ can be(CH₂)_(n)OR^(b) and R⁴ can be OR^(b); or

R³ can be (CH₂)_(n)OR^(a) and R⁴ can be OR^(b).

R¹ can be (CH₂)_(n)OR^(b) and R² can be OR^(a); or R¹ can be(CH₂)_(n)OR^(b) and R² can be OR^(b); or R¹ can be (CH₂)_(n)OR^(a) andR² can be OR^(b).

R³ can be (CH₂)_(n)OR^(b) and R⁹ can be OR^(a).

R³ and R⁹ can be cis, or R³ and R⁹ can be trans.

R³ can be (CH₂)_(n)OR^(b) and R⁹ can be OR^(b); or R³ can be(CH₂)_(n)OR^(b) and R⁹ can be OR^(a); or R³ can be (CH₂)_(n)OR^(a) andR⁹ can be OR^(b).

R¹ can be (CH₂)_(n)OR^(b) and R³ can be OR^(a).

R¹ and R³ can be cis, or R¹ and R³ can be trans.

R³ can be OR^(a) and R⁹ can be (CH₂)_(n)OR^(b).

R¹ can be OR^(a) and R³ can be (CH₂)_(n)OR^(b).

In other preferred embodiments the ribose is replaced with a piperazinescaffold, and X is N(CO)R⁷ or NR⁷, Y is NR⁸, and Z is CR¹¹R¹².

R¹ can be (CH₂)_(n)OR^(b) and R³ can be OR^(a).

R¹ and R³ can be cis or R¹ and R³ can be trans.

n can be 1.

R¹ can be (CH₂)_(n)OR^(b) and R³ can be OR^(b); or R¹ can be(CH₂)_(n)OR^(a) and R³ can be OR^(b).

A can be O or S, preferably S.

R⁷ can be can be T-R^(d), in which T is a tether having one or morelinking groups, wherein at least one of the linking groups is cleavable;and R^(d) can be H or a ligand, e.g., R^(d) can be chosen from a folicacid radical; a cholesterol radical; a carbohydrate radical; a vitamin Aradical; a vitamin E radical; a vitamin K radical. Preferably, R^(d) isa cholesterol radical.

R⁸ can be CH₃.

R¹ can be OR^(a) and R³ can be (CH₂)_(n)OR^(b).

In other preferred embodiments the ribose is replaced with a morpholinoscaffold, and X is N(CO)R⁷ or NR⁷, Y is O, and Z is CR¹¹R¹².

R¹ can be (CH₂)_(n)OR^(b) and R³ can be OR^(a).

R¹ and R³ can be cis, or R¹ and R³ can be trans.

n can be 1.

R¹ can be (CH₂)_(n)OR^(b) and R³ can be OR^(b); of R¹ can be(CH₂)_(n)OR^(a) and R³ can be OR^(b).

A can be O or S.

R⁷ can be can be T-R^(d), in which T is a tether having one or morelinking groups, wherein at least one of the linking groups is cleavable;and R^(d) can be H or a ligand, e.g., R^(d) can be chosen from a folicacid radical; a cholesterol radical; a carbohydrate radical; a vitamin Aradical; a vitamin E radical; a vitamin K radical. Preferably, R^(d) isa cholesterol radical.

R⁸ can be CH₃.

R¹ can be OR^(a) and R³ can be (CH₂)_(n)OR^(b).

In other preferred embodiments the ribose is replaced with a decalinscaffold, and X is CH₂; Y is CR⁹R¹⁰; and Z is CR¹¹R¹²; and R⁵ and R¹¹together are C⁶ cycloalkyl.

R⁶ can be C(O)NHR⁷.

R¹² can be hydrogen.

R⁶ and R¹² can be trans.

R³ can be OR^(a) and R⁹ can be (CH₂)_(n)OR^(b).

R³ and R⁹ can be cis, or R³ and R⁹ can be trans.

n can be 1 or 2.

R³ can be OR^(b) and R⁹ can be (CH₂)_(n)OR^(b); or R³ can be OR^(b) andR⁹ can be (CH₂)_(n)OR^(a).

A can be O or S.

R⁷ can be can be T-R^(d), in which T is a tether having one or morelinking groups, wherein at least one of the linking groups is cleavable;and R^(d) can be H or a ligand, e.g., R^(d) can be chosen from a folicacid radical; a cholesterol radical; a carbohydrate radical; a vitamin Aradical; a vitamin E radical; a vitamin K radical. Preferably, R^(d) isa cholesterol radical.

In other preferred embodiments the ribose is replaced with adecalin/indane scafold, e.g., X is CH₂; Y is CR⁹R¹⁰; and Z is CR¹¹R¹²;and R⁵ and R¹¹ together are C⁵ cycloalkyl.

R⁶ can be CH₃.

R¹² can be hydrogen.

R⁶ and R¹² can be trans.

R³ can be OR^(a) and R⁹ can be (CH₂)_(n)OR^(b).

R³ and R⁹ can be cis, or R³ and R⁹ can be trans.

n can be 1 or 2.

R³ can be OR^(b) and R⁹ can be (CH₂)_(n)OR^(a); or R³ can be OR^(b) andR⁹ can be (CH₂)_(n)OR^(a).

A can be O or S.

R¹⁴ can be N(CH3)R⁷. R⁷ can be can be T-R^(d), in which T is a tetherhaving one or more linking groups, wherein at least one of the linkinggroups is cleavable; and R^(d) can be H or a ligand, e.g., R^(d) can bechosen from a folic acid radical; a cholesterol radical; a carbohydrateradical; a vitamin A radical; a vitamin E radical; a vitamin K radical.Preferably, R^(d) is a cholesterol radical.

In another aspect, this invention features an iRNA agent comprising afirst strand and a second strand, wherein at least one one subunithaving a formula (II) is incorporated into at least one of said strands:

X is N(CO)R⁷ or NR⁷;

Each of R¹ and R² is, independently, OR^(a), OR^(b), (CH₂)_(n)OR^(a), or(CH₂)_(n)OR^(b), provided that one of R¹ and R² is OR^(a) or OR^(b) andthe other is (CH₂)_(n)OR^(a) or (CH₂)_(n)OR^(b) (when the RRMS isterminal, one of R¹ or R² will include R^(a) and one will include R^(b);when the RRMSS is internal, both R¹ and R² will each include an R^(b));further provided that preferably OR^(a) may only be present with(CH₂)_(n)OR^(b) and (CH₂)_(n)OR^(a) may only be present with OR^(b);

-   -   R⁷ is R⁷ can be can be T-R^(d), in which T is a tether having        one or more linking groups, wherein at least one of the linking        groups is cleavable; and R^(d) can be H or a ligand, e.g., R^(d)        can be chosen from a folic acid radical; a cholesterol radical;        a carbohydrate radical; a vitamin A radical; a vitamin E        radical; a vitamin K radical. Preferably, R^(d) is a cholesterol        radical;

R⁸ is C₁-C₆ alkyl;

R¹³ is hydroxy, C₁-C₄ alkoxy, or halo;

R¹⁴ is NR^(c)R⁷;

Each of A and C is, independently, O or S;

B is OH, O⁻, or

R^(c) is H or C₁-C₆ alkyl; and

n is 1-4.

Embodiments can include one or more of the features described above.

In a further aspect, this invention features an iRNA agent having afirst strand and a second strand, wherein at least one subunit having aformula (I) or formula (II) is incorporated into at least one of saidstrands.

In one aspect, this invention features an iRNA agent having a firststrand and a second strand, wherein at least two subunits having aformula (I) and/or formula (II) are incorporated into at least one ofsaid strands.

In another aspect, this invention provides a method of making an iRNAagent described herein having a first strand and a second strand inwhich at least one subunit of formula (I) and/or (II) is incorporated inthe strands. The method includes contacting the first strand with thesecond strand.

In a further aspect, this invention provides a method of modulatingexpression of a target gene, the method includes administering an iRNAagent described herein having a first strand and a second strand inwhich at least one subunit of formula (I) and/or (II) is incorporated inthe strands. to a subject.

In one aspect, this invention features a pharmaceutical compositionhaving an iRNA agent described herein having a first strand and a secondstrand in which at least one subunit of formula (I) and/or (II) isincorporated in the strands and a pharmaceutically acceptable carrier.

RRMSs described herein may be incorporated into any double-strandedRNA-like molecule described herein, e.g., an iRNA agent. An iRNA agentmay include a duplex comprising a hybridized sense and antisense strand,in which the antisense strand and/or the sense strand may include one ormore of the RRMSs described herein. An RRMS can be introduced at one ormore points in one or both strands of a double-stranded iRNA agent. AnRRMS can be placed at or near (within 1, 2, or 3 positions) of the 3′ or5′ end of the sense strand or at near (within 2 or 3 positions of) the3′ end of the antisense strand. In some embodiments it is preferred tonot have an RRMS at or near (within 1, 2, or 3 positions of) the 5′ endof the antisense strand. An RRMS can be internal, and will preferably bepositioned in regions not critical for antisense binding to the target.

In an embodiment, an iRNA agent may have an RRMS at (or within 1, 2, or3 positions of) the 3′ end of the antisense strand. In an embodiment, aniRNA agent may have an RRMS at (or within 1, 2, or 3 positions of) the3′ end of the antisense strand and at (or within 1, 2, or 3 positionsof) the 3′ end of the sense strand. In an embodiment, an iRNA agent mayhave an RRMS at (or within 1, 2, or 3 positions of) the 3′ end of theantisense strand and an RRMS at the 5′ end of the sense strand, in whichboth ligands are located at the same end of the iRNA agent.

In certain embodiments, two ligands are tethered, preferably, one oneach strand and are hydrophobic moieties. While not wishing to be boundby theory, it is believed that pairing of the hydrophobic ligands canstabilize the iRNA agent via intermolecular van der Waals interactions.

In an embodiment, an iRNA agent may have an RRMS at (or within 1, 2, or3 positions of) the 3′ end of the antisense strand and an RRMS at the 5′end of the sense strand, in which both RRMSs may share the same ligand(e.g., cholic acid) via connection of their individual tethers toseparate positions on the ligand. A ligand shared between two proximalRRMSs is referred to herein as a “hairpin ligand.”

In other embodiments, an iRNA agent may have an RRMS at the 3′ end ofthe sense strand and an RRMS at an internal position of the sensestrand. An iRNA agent may have an RRMS at an internal position of thesense strand; or may have an RRMS at an internal position of theantisense strand; or may have an RRMS at an internal position of thesense strand and an RRMS at an internal position of the antisensestrand.

In preferred embodiments the iRNA agent includes a first and secondsequences, which are preferably two separate molecules as opposed to twosequences located on the same strand, have sufficient complementarity toeach other to hybridize (and thereby form a duplex region), e.g., underphysiological conditions, e.g., under physiological conditions but notin contact with a helicase or other unwinding enzyme.

It is preferred that the first and second sequences be chosen such thatthe ds iRNA agent includes a single strand or unpaired region at one orboth ends of the molecule. Thus, a ds iRNA agent contains first andsecond sequences, preferable paired to contain an overhang, e.g., one ortwo 5′ or 3′ overhangs but preferably a 3′ overhang of 2-3 nucleotides.Most embodiments will have a 3′ overhang. Preferred sRNA agents willhave single-stranded overhangs, preferably 3′ overhangs, of 1 orpreferably 2 or 3 nucleotides in length at each end. The overhangs canbe the result of one strand being longer than the other, or the resultof two strands of the same length being staggered. 5′ ends arepreferably phosphorylated.

Other modifications to sugars, bases, or backbones described herein canbe incorpoated into the iRNA agents.

The iRNA agents can take an architecture or structure described herein.The iRNA agents can be palindromic, or double targeting, as describedherein.

The iRNA agents can have a sequence such that a non-cannonical or otherthan cannonical Watson-Crick structure is formed between two monomers ofthe iRNA agent or between a strand of the iRNA agent and anothersequence, e.g., a target or off-target sequence, as is described herein.

The iRNA agent can be selected to target any of a broad spectrum ofgenes, including any of the genes described herein.

In a preferred embodiment the iRNA agent has an architecture(architecture refers to one or more of overall length, length of aduplex region, the presence, number, location, or length of overhangs,single strand versus double strand form) described herein. E.g., theiRNA agent can be less than 30 nucleotides in length, e.g., 21-23nucleotides. Preferably, the iRNA is 21 nucleotides in length and thereis a duplex region of about 19 pairs. In one embodiment, the iRNA is 21nucleotides in length, and the duplex region of the iRNA is 19nucleotides. In another embodiment, the iRNA is greater than 30nucleotides in length.

In some embodiment the duplex region of the iRNA agent will have,mismatches. Preferably it will have no more than 1, 2, 3, 4, or 5 bases,which do not form canonical Watson-Crick pairs or which do nothybridize. Overhangs are discussed in detail elsewhere herein but arepreferably about 2 nucleotides in length. The overhangs can becomplementary to the gene sequences being targeted or can be othersequence. TT is a preferred overhang sequence. The first and second iRNAagent sequences can also be joined, e.g., by additional bases to form ahairpin, or by other non-base linkers.

In addition to the RRMS-containing bases the iRNA agents describedherein can include nuclease resistant monomers (NRMs).

In another aspect, the invention features an iRNA agent to which isconjugated a lipophilic moiety, e.g., cholesterol, e.g., by conjugationto an RRMS of an iRNA agent. In a preferred embodiment, the lipophilicmoiety enhances entry of the iRNA agent into a cell. In a preferredembodiment, the cell is part of an organism, tissue, or cell line, e.g.,a primary cell line, immortalized cell line, or any type of cell linedisclosed herein. Thus, the conjugated iRNA agent an be used to silencea target gene in an organism, e.g., a mammal, e.g., a human, or tosilence a target gene in a cell line or in cells which are outside anorganism.

The lipophilic moiety can be chosen, for example, from the groupconsisting of a lipid, cholesterol, oleyl, retinyl, cholesterylresidues, cholic acid, adamantane acetic acid, 1-pyrene butyric acid,dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexylgroup, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecylgroup, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid,O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine. A preferredlipophilic moiety is cholesterol.

The iRNA agent can have a first strand and a second strand, wherein atleast one subunit having formula (I) or formula (II) is incorporatedinto at least one of the strands. The iRNA agent can have one or more ofany of the features described herein. For example, when the subunit isof formula (I), R^(d) can be cholesterol; X can be N(CO)R⁷ or NR⁷, Y canbe CR⁹R¹⁰, and Z can be absent, and R¹ can be (CH₂)_(n)OR^(b) and R³ canbe OR^(a); X can be N(CO)R⁷ or NR⁷, Y can be CR⁹R¹⁰, and Z can beCR¹¹R¹², and R⁹ can be (CH₂)_(n)OR^(b) and R¹⁰ can be OR^(a); X can beN(CO)R⁷ or NR⁷, Y can be NR⁸, and Z can be CR¹¹R¹², and R¹ can be(CH₂)_(n)OR^(b) and R³ can be OR^(a); X can be CH₂; Y can be CR⁹R¹⁰; andZ can be CR¹¹R¹², in which R⁶ can be C(O)NHR⁷; or X can be CH₂; Y can beCR⁹R¹⁰; and Z can be CR¹¹R¹², in which R¹¹ or R¹² can be C(O)NHR⁷ or R⁵and R¹¹ together can be C₅ or C₆ cycloalkyl substituted with N(CH3)R⁷.

In a preferred embodiment, the lipophilic moiety, e.g., a cholesterol,enhances entry of the iRNA agent into a synoviocyte, myocyte,keratinocyte, hepatocyte, leukocyte, endothelial cell (e.g., a kidneycell), B-cell, T-cell, epithelial cell, mesodermal cell, myeloid cell,neural cell, neoplastic cell, mast cell, or fibroblast cell. In certainaspects, a myocyte can be a smooth muscle cell or a cardiac myocyte, afibroblast cell can be a dermal fibroblast, and a leukocyte can be amonocyte. In another preferred embodiment, the cell can be from anadherent tumor cell line derived from a tissue, such as bladder, lung,breast, cervix, colon, pancreas, prostate, kidney, liver, skin, ornervous system (e.g., central nervous system).

In a preferred embodiment, the iRNA agent targets a protein tyrosinephosphatase (PTP-1B) gene or a MAP kinase gene, such as ERK1, ERK2,JNK1, JNK2, or p38. In a preferred embodiment, these iRNA agents areused to silence genes in a fibroblast cell.

In a preferred embodiment, the iRNA agent targets an MDR, Myc, Myb,c-Myc, N-Myc, L-Myc, c-Myb, a-Myb, b-Myb, v-Myb, cyclin D1, Cyclin D2,cyclin E, CDK4, cdc25A, CDK2, or CDK4 gene. In a preferred embodiment,these iRNA agents are used to silence genes in an epithelial cell ormesodermal cell.

In a preferred embodiment, the iRNA agent targets a G72 or DAAO gene. Ina preferred embodiment, these iRNA agents are used to silence genes in aneural cell.

In a preferred embodiment, the iRNA agent targets a gene of thetelomerase pathway, such as a TERT or TR/TERC. In a preferredembodiment, these iRNA agents are used to silence genes in akeratinocyte.

In a preferred embodiment, the iRNA agent targets an interleukin gene,such as IL-1, IL-2, IL-5, IL-8, IL-10, IL-15, IL-16, IL-17, or IL-18. Inanother preferred embodiment, the iRNA agent targets an interleukinreceptor gene, or a chromosomal translocation, such as BCR-ABL,TEL-AML-1, EWS-FLI1, EWS-ERG, TLS-FUS, PAX3-FKHR, or AML-ETO. In apreferred embodiment, these iRNA agents are used to silence genes in alymphoma or a leukemia cell.

In a preferred embodiment, the iRNA agent targets a GRB2 associatedbinding protein. In a preferred embodiment, these iRNA agents are usedto silence genes in a mast cell.

In a preferred embodiment, the iRNA agent targets a growth factor orgrowth factor receptor, such as a TGFbeta or TGFbeta Receptor; PDGF orPDGFR; VEGF or VEGFr1, VEGFr2, or VEGFr3; or IGF-1R, DAF-2, or InR. Inanother preferred embodiment, the iRNA agent targets PRL1, PRL2, PRL3,protein kinase C (PKC), PKC receptor, MDR1, TERT, TR/TERC, cyclin D1,NF-KappaB, REL-A, REL-B, PCNA, CHK-1, c-fos, jun, or BCL-2. In apreferred embodiment, these iRNA agents are used to silence genes in anadherent tumor cell line.

In a preferred embodiment, the iRNA agent targets an exogenous gene of agenetically modified cell. An exogenous gene can be, for example, aviral or bacterial gene that derives from an organism that has invadedor infected the cell, or the exogenous gene can be any gene introducedinto the cell by natural or artificial means, such as by a geneticrecombination event. An iRNA agent can target a viral gene, for example,such as a hepatitis viral gene (e.g., a gene of an HAV, HBV, or HCV).Alternatively, or in addition, the iRNA agent can silence a reportergene, such as GFP or beta galatosidase and the like. These iRNA agentscan be used to silence exogenous genes in an adherent tumor cell line.

In a preferred embodiment, the iRNA agent to which the lipophilic moietyis conjugated silences at least one gene, e.g., any gene describedherein, in any one of a number of cell lines including, but not limitedto, a 3T3, DLD2, THP1, Raw264.7, IC21, P388D1, U937, HL60, SEM-K2,WEHI-231, HB56, TIB55, Jurkat, J45.01, K562, EL4, LRMB, Bcl-1, BC-3,TF1, CTLL-2, C1R, Rat6, VERO, MRC5, CV1, Cos7, RPTE, A10, T24, J82,A549, A375, ARH-77, Calu1, SW480, SW620, SKOV3, SK-UT, CaCo2, A375,C8161, CCRF-CEM, MCF-7, MDA-MB-231, MOLT, mIMCD-3, NHDF, HeLa, HeLa-S3,Huh1, Huh4, Huh7, HUVEC, HASMC, HEKn, HEKa, MiaPaCell, Panc1, PC-3,LNCaP, HepG2, or U87 cell line. Cell lines are available from a varietyof sources known to those with skill in the art (see, e.g., the AmericanType Culture Collection (ATCC) (Manassus, Va.)).

In another aspect, the invention provides, methods of silencing a targetgene by providing an iRNA agent to which a lipophilic moiety isconjugated, e.g., a lipophilic conjugated iRNA agent described herein,to a cell. In a preferred embodiment the conjugated iRNA agent an beused to silence a target gene in an organism, e.g., a mammal, e.g., ahuman, or to silence a target gene in a cell line or in cells which areoutside an organism. In the case of a whole organism, the method can beused to silence a gene, e.g., a gene described herein, and treat acondition mediated by the gene. In the case of use on a cell which isnot part of an organism, e.g., a primary cell line, secondary cell line,tumor cell line, or transformed or immortalized cell line, the iRNAagent to which a lipophilic moiety is conjugated can be used to silencea gene, e.g., one described herein. Cells which are not part of a wholeorganism can be used in an initial screen to determine if an iRNA agentis effective in silencing a gene. A test in cells which are not part ofa whole organism can be followed by testing the iRNA agent in a wholeanimal. In preferred embodiments, the iRNA agent which is conjugated toa lipophilic moiety is administered to an organism, or contacted with acell which is not part of an organism, in the absence of (or in areduced amount of) other reagents that facilitate or enhance delivery,e.g., a compound which enhances transit through the cell membrane. (Areduced amount can be an amount of such reagent which is reduced incomparison to what would be needed to get an equal amount ofnonconjugated iRNA agent into the target cell). E.g., the iRNA agentwhich is conjugated to a lipophilic moiety is administered to anorganism, or contacted with a cell which is not part of an organism, inthe absence (or reduced amount) of: an additional lipophilic moiety; atransfection agent, e.g., concentrations of an ion or other substancewhich substantially alters cell permeability to an iRNA agent; atransfecting agent such as Lipofectamine™ (Invitrogen, Carlsbad,Calif.), Lipofectamine 2000™, TransIT-TKO™ (Mirus, Madison, Wis.),FuGENE 6 (Roche, Indianapolis, Ind.), polyethylenimine, X-tremeGENE Q2(Roche, Indianapolis, Ind.), DOTAP, DOSPER, Metafectene™ (Biontex,Munich, Germany), and the like.

In a preferred embodiment the iRNA agent is suitable for delivery to acell in vivo, e.g., to a cell in an organism. In another aspect, theiRNA agent is suitable for delivery to a cell in vitro, e.g., to a cellin a cell line.

An iRNA agent to which a lipophilic moiety is attached can target anygene described herein and can be delivered to any cell type describedherein, e.g., a cell type in an organism, tissue, or cell line. Deliveryof the iRNA agent can be in vivo, e.g., to a cell in an organism, or invitro, e.g., to a cell in a cell line.

In another aspect, the invention provides compositions of iRNA agentsdescribed herein, and in particular compositions of an iRNA agent towhich a lipophilic moiety is conjugated, e.g., a lipophilic conjugatediRNA agent described herein. In a preferred embodiment the compositionis a pharmaceutically acceptable composition.

In preferred embodiments, the composition, e.g., pharmaceuticallyacceptable composition, is free of, has a reduced amount of, or isessentially free of other reagents that facilitate or enhance delivery,e.g., compounds which enhance transit through the cell membrane. (Areduced amount can be an amount of such reagent which is reduced incomparison to what would be needed to get an equal amount ofnonconjugated iRNA agent into the target cell). E.g., the composition isfree of, has a reduced amount of, or is essentially free of: anadditional lipophilic moiety; a transfection agent, e.g., concentrationsof an ion or other substance which substantially alters cellpermeability to an iRNA agent; a transfecting agent such asLipofectamine™ (Invitrogen, Carlsbad, Calif.), Lipofectamine 2000™,TransIT-TKO™ (Mirus, Madison, Wis.), FuGENE 6 (Roche, Indianapolis,Ind.), polyethylenimine, X-tremeGENE Q2 (Roche, Indianapolis, Ind.),DOTAP, DOSPER, Metafectene™ (Biontex, Munich, Germany), and the like.

In a preferred embodiment the composition is suitable for delivery to acell in vivo, e.g., to a cell in an organism. In another aspect, theiRNA agent is suitable for delivery to a cell in vitro, e.g., to a cellin a cell line.

The RRMS-containing iRNA agents can be used in any of the methodsdescribed herein, e.g., to target any of the genes described herein orto treat any of the disorders described herein. They can be incorporatedinto any of the formulations, modes of delivery, delivery modalities,kits or preparations, e.g., pharmaceutical preparations, describedherein. E.g, a kit which inlcudes one or more of the iRNA aentsdescribed herein, a sterile container in which the iRNA agent isdiscolsed, and instructions for use.

The methods and compositions of the invention, e.g., the RRSM-containingiRNA agents described herein, can be used with any of the iRNA agentsdescribed herein. In addition, the methods and compositions of theinvention can be used for the treatment of any disease or disorderdescribed herein, and for the treatment of any subject, e.g., anyanimal, any mammal, such as any human.

The methods and compositions of the invention, e.g., the theRRMS-containing iRNA agents described herein, can be used with anydosage and/or formulation described herein, as well as with any route ofadministration described herein.

The non-ribose scaffolds, as well as monomers and dimers of the RRMSsdescribed herein are within the invention.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features andadvantages of the invention will be apparent from the description anddrawings, and from the claims. This application incorporates all citedreferences, patents, and patent applications by references in theirentirety for all purposes.

DESCRIPTION OF DRAWINGS

FIG. 1 a general synthetic scheme for incorporation of RRMS monomersinto an oligonucleotide.

FIG. 2A is a list of substituents that may be present on silicon inOFG¹.

FIG. 2B is a list of substituents that may be present on theC2′-orthoester group.

FIG. 3 is list of representative RRMS cyclic carriers. Panel 1 showspyrroline-based RRMSs; panel 2 shows 3-hydroxyproline-based RRMSs; panel3 shows piperidine-based RRMSs; panel 4 shows morpholine andpiperazine-based RRMSs; and panel 5 shows decalin-based RRMSs. R1 issuccinate or phosphoramidate and R2 is H or a conjugate ligand.

FIG. 4 is a general reaction scheme for 3′ conjugation of peptide intoiRNA.

FIG. 5 is a general reaction scheme for 5′ conjugation of peptide intoiRNA.

FIG. 6 is a general reaction scheme for the synthesis of aza-peptides.

FIG. 7 is a general reaction scheme for the synthesis of N-methyl aminoacids and peptides.

FIG. 8 is a general reaction scheme for the synthesis of β-methyl aminoacids and Ant and Tat peptides.

FIG. 9 is a general reaction scheme for the synthesis of Ant and Tatoligocarbamates.

FIG. 10 is a a general reaction scheme for the synthesis of Ant and Tatoligoureas.

FIG. 11 is a schematic representation of peptide carriers.

DETAILED DESCRIPTION

Double-stranded (dsRNA) directs the sequence-specific silencing of mRNAthrough a process known as RNA interference (RNAi). The process occursin a wide variety of organisms, including mammals and other vertebrates.

It has been demonstrated that 21-23 nt fragments of dsRNA aresequence-specific mediators of RNA silencing, e.g., by causing RNAdegradation. While not wishing to be bound by theory, it may be that amolecular signal, which may be merely the specific length of thefragments, present in these 21-23 nt fragments recruits cellular factorsthat mediate RNAi. Described herein are methods for preparing andadministering these 21-23 nt fragments, and other iRNAs agents, andtheir use for specifically inactivating gene function. The use of iRNAsagents (or recombinantly produced or chemically synthesizedoligonucleotides of the same or similar nature) enables the targeting ofspecific mRNAs for silencing in mammalian cells. In addition, longerdsRNA agent fragments can also be used, e.g., as described below.

Although, in mammalian cells, long dsRNAs can induce the interferonresponse which is frequently deleterious, sRNAs do not trigger theinterferon response, at least not to an extent that is deleterious tothe cell and host. In particular, the length of the iRNA agent strandsin an sRNA agent can be less than 31, 30, 28, 25, or 23 nt, e.g.,sufficiently short to avoid inducing a deleterious interferon response.Thus, the administration of a composition of sRNA agent (e.g.,formulated as described herein) to a mammalian cell can be used tosilence expression of a target gene while circumventing the interferonresponse. Further, use of a discrete species of iRNA agent can be usedto selectively target one allele of a target gene, e.g., in a subjectheterozygous for the allele.

Moreover, in one embodiment, a mammalian cell is treated with an iRNAagent that disrupts a component of the interferon response, e.g., doublestranded RNA (dsRNA)-activated protein kinase PKR. Such a cell can betreated with a second iRNA agent that includes a sequence complementaryto a target RNA and that has a length that might otherwise trigger theinterferon response.

As used herein, a “subject” refers to a mammalian organism undergoingtreatment for a disorder mediated by unwanted target gene expression.The subject can be any mammal, such as a cow, horse, mouse, rat, dog,pig, goat, or a primate. In the preferred embodiment, the subject is ahuman.

Because iRNA agent mediated silencing persists for several days afteradministering the iRNA agent composition, in many instances, it ispossible to administer the composition with a frequency of less thanonce per day, or, for some instances, only once for the entiretherapeutic regimen. For example, treatment of some cancer cells may bemediated by a single bolus administration, whereas a chronic viralinfection may require regular administration, e.g., once per week oronce per month.

A number of exemplary routes of delivery are described that can be usedto administer an iRNA agent to a subject. In addition, the iRNA agentcan be formulated according to an exemplary method described herein.

Ligand-Conjugated Monomer Subunits and Monomers for OligonucleotideSynthesis

Definitions

The term “halo” refers to any radical of fluorine, chlorine, bromine oriodine.

The term “alkyl” refers to a hydrocarbon chain that may be a straightchain or branched chain, containing the indicated number of carbonatoms. For example, C₁-C₁₂ alkyl indicates that the group may have from1 to 12 (inclusive) carbon atoms in it. The term “haloalkyl” refers toan alkyl in which one or more hydrogen atoms are replaced by halo, andincludes alkyl moieties in which all hydrogens have been replaced byhalo (e.g., perfluoroalkyl). Alkyl and haloalkyl groups may beoptionally inserted with O, N, or S. The terms “aralkyl” refers to analkyl moiety in which an alkyl hydrogen atom is replaced by an arylgroup. Aralkyl includes groups in which more than one hydrogen atom hasbeen replaced by an aryl group. Examples of “aralkyl” include benzyl,9-fluorenyl, benzhydryl, and trityl groups.

The term “alkenyl” refers to a straight or branched hydrocarbon chaincontaining 2-8 carbon atoms and characterized in having one or moredouble bonds. Examples of a typical alkenyl include, but not limited to,allyl, propenyl, 2-butenyl, 3-hexenyl and 3-octenyl groups. The term“alkynyl” refers to a straight or branched hydrocarbon chain containing2-8 carbon atoms and characterized in having one or more triple bonds.Some examples of a typical alkynyl are ethynyl, 2-propynyl, and3-methylbutynyl, and propargyl. The sp² and sp³ carbons may optionallyserve as the point of attachment of the alkenyl and alkynyl groups,respectively.

The terms “alkylamino” and “dialkylamino” refer to —NH(alkyl) and —N(alkyl)₂ radicals respectively. The term “aralkylamino” refers to a—NH(aralkyl) radical. The term “alkoxy” refers to an —O-alkyl radical,and the terms “cycloalkoxy” and “aralkoxy” refer to an —O-cycloalkyl andO-aralkyl radicals respectively. The term “siloxy” refers to a R₃SiO—radical. The term “mercapto” refers to an SH radical. The term“thioalkoxy” refers to an —S-alkyl radical.

The term “alkylene” refers to a divalent alkyl (i.e., —R—), e.g., —CH₂—,—CH₂CH₂—, and —CH₂CH₂CH₂—. The term alkenylene refers to a divalentalkenyl (e.g., —CH₂CH═CH—, polyalkenyl). The term alkynylene refers to adivalent alkynyl (e.g., propargyl, polyalkynyl). The term“alkylenedioxo” refers to a divalent species of the structure —O—R—O—,in which R represents an alkylene.

The term “aryl” refers to an aromatic monocyclic, bicyclic, or tricyclichydrocarbon ring system, wherein any ring atom can be substituted.Examples of aryl moieties include, but are not limited to, phenyl,naphthyl, anthracenyl, and pyrenyl.

The term “cycloalkyl” as employed herein includes saturated cyclic,bicyclic, tricyclic, or polycyclic hydrocarbon groups having 3 to 12carbons, wherein any ring atom can be substituted. The cycloalkyl groupsherein described may also contain fused rings. Fused rings are ringsthat share a common carbon-carbon bond or a common carbon atom (e.g.,spiro-fused rings). Examples of cycloalkyl moieties include, but are notlimited to, cyclohexyl, adamantyl, and norbornyl.

The term “heterocyclyl” refers to a nonaromatic 3-10 memberedmonocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic ringsystem having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms ifbicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms selectedfrom O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms ofN, O, or S if monocyclic, bicyclic, or tricyclic, respectively), whereinany ring atom can be substituted. The heterocyclyl groups hereindescribed may also contain fused rings. Fused rings are rings that sharea common carbon-carbon bond or a common carbon atom (e.g., spiro-fusedrings). Examples of heterocyclyl include, but are not limited totetrahydrofuranyl, tetrahydropyranyl, piperidinyl, morpholino,pyrrolinyl and pyrrolidinyl.

The term “cycloalkenyl” as employed herein includes partiallyunsaturated, nonaromatic, cyclic, bicyclic, tricyclic, or polycyclichydrocarbon groups having 5 to 12 carbons, preferably 5 to 8 carbons,wherein any ring atom can be substituted. The cycloalkenyl groups hereindescribed may also contain fused rings. Fused rings are rings that sharea common carbon-carbon bond or a common carbon atom (e.g., spiro-fusedrings). Examples of cycloalkenyl moieties include, but are not limitedto cyclohexenyl, cyclohexadienyl, or norbornenyl.

The term “heterocycloalkenyl” refers to a partially saturated,nonaromatic 5-10 membered monocyclic, 8-12 membered bicyclic, or 11-14membered tricyclic ring system having 1-3 heteroatoms if monocyclic, 1-6heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, saidheteroatoms selected from O, N, or S (e.g., carbon atoms and 1-3, 1-6,or 1-9 heteroatoms of N, O, or S if monocyclic, bicyclic, or tricyclic,respectively), wherein any ring atom can be substituted. Theheterocycloalkenyl groups herein described may also contain fused rings.Fused rings are rings that share a common carbon-carbon bond or a commoncarbon atom (e.g., spiro-fused rings). Examples of heterocycloalkenylinclude but are not limited to tetrahydropyridyl and dihydropyran.

The term “heteroaryl” refers to an aromatic 5-8 membered monocyclic,8-12 membered bicyclic, or 11-14 membered tricyclic ring system having1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9heteroatoms if tricyclic, said heteroatoms selected from O, N, or S(e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S ifmonocyclic, bicyclic, or tricyclic, respectively), wherein any ring atomcan be substituted. The heteroaryl groups herein described may alsocontain fused rings that share a common carbon-carbon bond.

The term “oxo” refers to an oxygen atom, which forms a carbonyl whenattached to carbon, an N-oxide when attached to nitrogen, and asulfoxide or sulfone when attached to sulfur.

The term “acyl” refers to an alkylcarbonyl, cycloalkylcarbonyl,arylcarbonyl, heterocyclylcarbonyl, or heteroarylcarbonyl substituent,any of which may be further substituted by substituents.

The term “substituents” refers to a group “substituted” on an alkyl,cycloalkyl, alkenyl, alkynyl, heterocyclyl, heterocycloalkenyl,cycloalkenyl, aryl, or heteroaryl group at any atom of that group.Suitable substituents include, without limitation, alkyl, alkenyl,alkynyl, alkoxy, halo, hydroxy, cyano, nitro, amino, SO₃H, sulfate,phosphate, perfluoroalkyl, perfluoroalkoxy, methylenedioxy,ethylenedioxy, carboxyl, oxo, thioxo, imino (alkyl, aryl, aralkyl),S(O)_(n)alkyl (where n is 0-2), S(O)_(n) aryl (where n is 0-2), S(O)_(n)heteroaryl (where n is 0-2), S(O)_(n) heterocyclyl (where n is 0-2),amine (mono-, di-, alkyl, cycloalkyl, aralkyl, heteroaralkyl, andcombinations thereof), ester (alkyl, aralkyl, heteroaralkyl), amide(mono-, di-, alkyl, aralkyl, heteroaralkyl, and combinations thereof),sulfonamide (mono-, di-, alkyl, aralkyl, heteroaralkyl, and combinationsthereof), unsubstituted aryl, unsubstituted heteroaryl, unsubstitutedheterocyclyl, and unsubstituted cycloalkyl. In one aspect, thesubstituents on a group are independently any one single, or any subsetof the aforementioned substituents.

The terms “adeninyl, cytosinyl, guaninyl, thyminyl, and uracilyl” andthe like refer to radicals of adenine, cytosine, guanine, thymine, anduracil.

A “protected” moiety refers to a reactive functional group, e.g., ahydroxyl group or an amino group, or a class of molecules, e.g., sugars,having one or more functional groups, in which the reactivity of thefunctional group is temporarily blocked by the presence of an attachedprotecting group. Protecting groups useful for the monomers and methodsdescribed herein can be found, e.g., in Greene, T. W., Protective Groupsin Organic Synthesis (John Wiley and Sons: New York), 1981, which ishereby incorporated by reference.

General

An RNA agent, e.g., an iRNA agent, containing a preferred, butnonlimiting ligand-conjugated monomer subunit is presented as formula(II) below and in the scheme in FIG. 1. The carrier (also referred to insome embodiments as a “linker”) can be a cyclic or acyclic moiety andincludes two “backbone attachment points” (e.g., hydroxyl groups) and aligand. The ligand can be directly attached (e.g., conjugated) to thecarrier or indirectly attached (e.g., conjugated) to the carrier by anintervening tether (e.g., an acyclic chain of one or more atoms; or anucleobase, e.g., a naturally occurring nucleobase optionally having oneor more chemical modifications, e.g., an unusual base; or a universalbase). The carrier therefore also includes a “ligand or tetheringattachment point” for the ligand and tether/tethered ligand,respectively.

The ligand-conjugated monomer subunit may be the 5′ or 3′ terminalsubunit of the RNA molecule, i.e., one of the two “W” groups may be ahydroxyl group, and the other “W” group may be a chain of two or moreunmodified or modified ribonucleotides. Alternatively, theligand-conjugated monomer subunit may occupy an internal position, andboth “W” groups may be one or more unmodified or modifiedribonucleotides. More than one ligand-conjugated monomer subunit may bepresent in a RNA molecule, e.g., an iRNA agent. Preferred positions forinclusion of a tethered ligand-conjugated monomer subunits, e.g., one inwhich a lipophilic moiety, e.g., cholesterol, is tethered to the carrierare at the 3′ terminus, the 5′ terminus, or an internal position of thesense strand.

The modified RNA molecule of formula (II) can be obtained usingoligonucleotide synthetic methods known in the art. In a preferredembodiment, the modified RNA molecule of formula (II) can be prepared byincorporating one or more of the corresponding monomer compounds (see,e.g., A, B, and C below and in the scheme in FIG. 1) into a growingsense or antisense strand, utilizing, e.g., phosphoramidite orH-phosphonate coupling strategies.

The monomers, e.g., a ligand-conjugated monomer, generally include twodifferently functionalized hydroxyl groups (OFG¹ and OFG²), which arelinked to the carrier molecule (see A below and in FIG. 1), and aligand/tethering attachment point. As used herein, the term“functionalized hydroxyl group” means that the hydroxylproton has beenreplaced by another substituent. As shown in representative structures Band C below and in FIG. 1, one hydroxyl group (OFG¹) on the carrier isfunctionalized with a protecting group (PG). The other hydroxyl group(OFG²) can be functionalized with either (1) a liquid or solid phasesynthesis support reagent (solid circle) directly or indirectly througha linker, L, as in B, or (2) a phosphorus-containing moiety, e.g., aphosphoramidite as in C. The tethering attachment point may be connectedto a hydrogen atom, a suitable protecting group, a tether, or a tetheredligand at the time that the monomer is incorporated into the growingsense or antisense strand (see variable “R” in A below). Thus, thetethered ligand can be, but need not be attached to the monomer at thetime that the monomer is incorporated into the growing strand. Incertain embodiments, the tether, the ligand or the tethered ligand maybe linked to a “precursor” ligand-conjugated monomer subunit after a“precursor” ligand-conjugated monomer subunit has been incorporated intothe strand. The wavy line used below (and elsewhere herein) refers to aconnection, and can represent a direct bond between the moiety and theattachment point or a tethering molecule which is interposed between themoiety and the attachment point. Directly tethered means the moiety isbound directly to the attachment point. Indirectly tethered means thatthere is a tether molecule interposed between the attachment point andthe moiety.

The (OFG¹) protecting group may be selected as desired, e.g., from T. W.Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis, 2d.Ed., John Wiley and Sons (1991). The protecting group is preferablystable under amidite synthesis conditions, storage conditions, andoligonucleotide synthesis conditions. Hydroxyl groups, —OH, arenucleophilic groups (i.e., Lewis bases), which react through the oxygenwith electrophiles (i.e., Lewis acids). Hydroxyl groups in which thehydrogen has been replaced with a protecting group, e.g., atriarylmethyl group or a trialkylsilyl group, are essentially unreactiveas nucleophiles in displacement reactions. Thus, the protected hydroxylgroup is useful in preventing e.g., homocoupling of compoundsexemplified by structure C during oligonucleotide synthesis. In someembodiments, a preferred protecting group is the dimethoxytrityl group.In other embodiments, a preferred protecting group is a silicon-basedprotecting group having the formula below:

X5′, X5″, and X5′″ can be selected from substituted or unsubstitutedalkyl, cycloalkyl, aryl, araklyl, heteroaryl, alkoxy, cycloalkoxy,aralkoxy, aryloxy, heteroaryloxy, or siloxy (i.e., R₃SiO—, the three “R”groups can be any combination of the above listed groups). X^(5′),X^(5″), and X^(5′″) may all be the same or different; also contemplatedis a combination in which two of X^(5′), X^(5″), and X^(5′″) areidentical and the third is different. In certain embodiments X^(5′),X^(5″), and X^(5′″) include at least one alkoxy or siloxy groups and maybe any one of the groups listed in FIG. 2A, a preferred combinationincludes X^(5′), X^(5″)=trimethylsiloxy andX^(5′″)=1,3-(triphenylmethoxy)-2-propoxy or cyclododecyloxy.

Other preferred combinations of X^(5′), X^(5″), and X^(5′″) includethose that result in OFG¹ groups that meet the deprotection andstability criteria delineated below. The group is preferably stableunder amidite synthesis conditions, storage conditions, andoligonucleotide synthesis conditions. Rapid removal, i.e., less than oneminute, of the silyl group from e.g., a support-bound oligonucleotide isdesirable because it can reduce synthesis times and thereby reduceexposure time of the growing oligonucleotide chain to the reagents.Oligonucleotide synthesis can be improved if the silyl protecting groupis visible during deprotection, e.g., from the addition of a chromophoresilyl substituent.

Selection of silyl protecting groups can be complicated by the competingdemands of the essential characteristics of stability and facileremoval, and the need to balance these competitive goals. Mostsubstituents that increase stability can also increase the reaction timerequired for removal of the silyl group, potentially increasing thelevel of difficulty in removal of the group.

The addition of alkoxy and siloxy substituents to OFG¹silicon-containing protecting groups increases the susceptibility of theprotecting groups to fluoride cleavage of the silylether bonds.Increasing the steric bulk of the substituents preserves stability whilenot decreasing fluoride lability to an equal extent. An appropriatebalance of substituents on the silyl group makes a silyl ether a viablenucleoside protecting group.

Candidate OFG¹ silicon-containing protecting groups may be tested byexposing a tetrahydrofuran solution of a preferred carrier bearing thecandidate OFG¹ group to five molar equivalents of tetrahydrofuran atroom temperature. The reaction time may be determined by monitoring thedisappearance of the starting material by thin layer chromatography.

When the OFG² in B includes a linker, e.g., a relatively long organiclinker, connected to a soluble or insoluble support reagent, solution orsolid phase synthesis techniques can be employed to build up a chain ofnatural and/or modified ribonucleotides once OFG¹ is deprotected andfree to react as a nucleophile with another nucleoside or monomercontaining an electrophilic group (e.g., an amidite group).Alternatively, a natural or modified ribonucleotide oroligoribonucleotide chain can be coupled to monomer C via an amiditegroup or H-phosphonate group at OFG². Subsequent to this operation, OFG¹can be deblocked, and the restored nucleophilic hydroxyl group can reactwith another nucleoside or monomer containing an electrophilic group. R′can be substituted or unsubstituted alkyl or alkenyl. In preferredembodiments, R′ is methyl, allyl or 2-cyanoethyl. R″ may a C₁-C₁₀ alkylgroup, preferably it is a branched group containing three or morecarbons, e.g., isopropyl.

OFG² in B can be hydroxyl functionalized with a linker, which in turncontains a liquid or solid phase synthesis support reagent at the otherlinker terminus. The support reagent can be any support medium that cansupport the monomers described herein. The monomer can be attached to aninsoluble support via a linker, L, which allows the monomer (and thegrowing chain) to be solubilized in the solvent in which the support isplaced. The solubilized, yet immobilized, monomer can react withreagents in the surrounding solvent; unreacted reagents and solubleby-products can be readily washed away from the solid support to whichthe monomer or monomer-derived products is attached. Alternatively, themonomer can be attached to a soluble support moiety, e.g., polyethyleneglycol (PEG) and liquid phase synthesis techniques can be used to buildup the chain. Linker and support medium selection is within skill of theart. Generally the linker may be —C(O)(CH₂)_(q)C(O)—, or—C(O)(CH₂)_(q)S—, in which q can be 0, 1, 2, 3, or 4; preferably, it isoxalyl, succinyl or thioglycolyl. Standard control pore glass solidphase synthesis supports can not be used in conjunction with fluoridelabile 5′ silyl protecting groups because the glass is degraded byfluoride with a significant reduction in the amount of full-lengthproduct. Fluoride-stable polystyrene based supports or PEG arepreferred.

The ligand/tethering attachment point can be any divalent, trivalent,tetravalent, pentavalent or hexavalent atom. In some embodiments,ligand/tethering attachment point can be a carbon, oxygen, nitrogen orsulfur atom. For example, a ligand/tethering attachment point precursorfunctional group can have a nucleophilic heteroatom, e.g., —SH, —NH₂,secondary amino, ONH₂, or NH₂NH₂. As another example, theligand/tethering attachment point precursor functional group can be anolefin, e.g., —CH═CH₂, and the precursor functional group can beattached to a ligand, a tether, or tethered ligand using, e.g.,transition metal catalyzed carbon-carbon (for example olefin metathesis)processes or cycloadditions (e.g., Diels-Alder). As a further example,the ligand/tethering attachment point precursor functional group can bean electrophilic moiety, e.g., an aldehyde. When the carrier is a cycliccarrier, the ligand/tethering attachment point can be an endocyclic atom(i.e., a constituent atom in the cyclic moiety, e.g., a nitrogen atom)or an exocyclic atom (i.e., an atom or group of atoms attached to aconstituent atom in the cyclic moiety).

The carrier can be any organic molecule containing attachment points forOFG¹, OFG², and the ligand. In certain embodiments, carrier is a cyclicmolecule and may contain heteroatoms (e.g., O, N or S). E.g., carriermolecules may include aryl (e.g., benzene, biphenyl, etc.), cycloalkyl(e.g., cyclohexane, cis or trans decalin, etc.), or heterocyclyl(piperazine, pyrrolidine, etc.). In other embodiments, the carrier canbe an acyclic moiety, e.g., based on serinol. Any of the above cyclicsystems may include substituents in addition to OFG¹, OFG², and theligand.

Sugar-Based Monomers

In some embodiments, the carrier molecule is an oxygen containingheterocycle. Preferably the carrier is a ribose sugar as shown instructure LCM-I. In this embodiment, the monomer, e.g., aligand-conjugated monomer is a nucleoside.

“B” represents a nucleobase, e.g., a naturally occurring nucleobaseoptionally having one or more chemical modifications, e.g., and unusualbase; or a universal base.

As used herein, an “unusual” nucleobase can include any one of thefollowing:

-   2-methyladeninyl,-   N6-methyladeninyl,-   2-methylthio-N-6-methyladeninyl,-   N6-isopentenyladeninyl,-   2-methylthio-N-6-isopentenyladeninyl,-   N6-(cis-hydroxyisopentenyl)adeninyl,-   2-methylthio-N6-(cis-hydroxyisopentenyl)adeninyl,-   N6-glycinylcarbamoyladeninyl,-   N6-threonylcarbamoyladeninyl,-   2-methylthio-N6-threonyl carbamoyladeninyl,-   N6-methyl-N6-threonylcarbamoyladeninyl,-   N6-hydroxynorvalylcarbamoyladeninyl,-   2-methylthio-N6-hydroxynorvalyl carbamoyladeninyl,-   N6,N6-dimethyladeninyl,-   3-methylcytosinyl,-   5-methylcytosinyl,-   2-thiocytosinyl,-   5-formylcytosinyl,

-   N4-methylcytosinyl,-   5-hydroxymethylcytosinyl,-   1-methylguaninyl,-   N2-methylguaninyl,-   7-methylguaninyl,-   N2,N2-dimethylguaninyl,

-   N2,7-dimethylguaninyl,-   N2,N2,7-trimethylguaninyl,-   1-methylguaninyl,-   7-cyano-7-deazaguaninyl,-   7-aminomethyl-7-deazaguaninyl,-   pseudouracilyl,-   dihydrouracilyl,-   5-methyluracilyl,-   1-methylpseudouracilyl,-   2-thiouracilyl,-   4-thiouracilyl,-   2-thiothyminyl-   5-methyl-2-thiouracilyl,-   3-(3-amino-3-carboxypropyl)uracilyl,-   5-hydroxyuracilyl,-   5-methoxyuracilyl,-   uracilyl 5-oxyacetic acid,-   uracilyl 5-oxyacetic acid methyl ester,-   5-(carboxyhydroxymethyl)uracilyl,-   5-(carboxyhydroxymethyl)uracilyl methyl ester,-   5-methoxycarbonylmethyluracilyl,-   5-methoxycarbonylmethyl-2-thiouracilyl,-   5-aminomethyl-2-thiouracilyl,-   5-methylaminomethyluracilyl,-   5-methylaminomethyl-2-thiouracilyl,-   5-methylaminomethyl-2-selenouracilyl,-   5-carbamoylmethyluracilyl,-   5-carboxymethylaminomethyluracilyl,-   5-carboxymethylaminomethyl-2-thiouracilyl,-   3-methyluracilyl,-   1-methyl-3-(3-amino-3-carboxypropyl) pseudouracilyl,-   5-carboxymethyluracilyl,-   5-methyldihydrouracilyl, or-   3-methylpseudouracilyl.

A universal base can form base pairs with each of the natural DNA/RNAbases, exhibiting relatively little discrimination between them. Ingeneral, the universal bases are non-hydrogen bonding, hydrophobic,aromatic moieties which can stabilize e.g., duplex RNA or RNA-likemolecules, via stacking interactions. A universal base can also includehydrogen bonding substituents. As used herein, a “universal base” caninclude anthracenes, pyrenes or any one of the following:

In some embodiments, B can form part of a tether that connects a ligandto the carrier. For example, the tether can beB—CH═CH—C(O)NH—(CH₂)₅—NHC(O)-LIGAND. In a preferred embodiment, thedouble bond is trans, and the ligand is a substituted or unsubstitutedcholesterolyl radical (e.g., attached through the D-ring side chain orthe C-3 hydroxyl); an aralkyl moiety having at least one sterogeniccenter and at least one substituent on the aryl portion of the aralkylgroup; or a nucleobase. In certain embodiments, B, in the tetherdescribed above, is uracilyl or a universal base, e.g., an aryl moiety,e.g., phenyl, optionally having additional substituents, e.g., one ormore fluoro groups. B can be substituted at any atom with the remainderof the tether.

X² can include “oxy” or “deoxy” substituents in place of the 2′-OH; orbe a ligand or a tethered ligand.

Examples of “oxy”-substituents include alkoxy or aryloxy (OR, e.g., R=H,alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, sugar, or protectinggroup); polyethyleneglycols (PEG), O(CH₂CH₂O)_(n)CH₂CH₂OR; “locked”nucleic acids (LNA) in which the 2′ hydroxyl is connected, e.g., by amethylene bridge, to the 4′ carbon of the same ribose sugar; O-PROTECTEDAMINE (AMINE=NH₂; alkylamino, dialkylamino, heterocyclyl, arylamino,diaryl amino, heteroaryl amino, or diheteroaryl amino, ethylene diamine,polyamino) and aminoalkoxy, O(CH₂)_(n)PROTECTED AMINE, (e.g., AMINE=NH₂;alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino,heteroaryl amino, or diheteroaryl amino, ethylene diamine, polyamino),and orthoester. Amine protecting groups can include formyl, amido,benzyl, allyl, etc.

Preferred orthoesters have the general formula J. The groups R³¹ and R³²may be the same or different and can be any combination of the groupslisted in FIG. 2B. A preferred orthoester is the “ACE” group, shownbelow as structure K.

“Deoxy” substituents include hydrogen (i.e. deoxyribose sugars); halo(e.g., fluoro); protected amino (e.g. NH₂; alkylamino, dialkylamino,heterocyclyl, arylamino, diaryl amino, heteroaryl amino, diheteroarylamino, or amino acid in which all amino are protected); fully protectedpolyamino (e.g., NH(CH₂CH₂NH)_(n)CH₂CH₂-AMINE, wherein AMINE=NH₂;alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino,heteroaryl amino, or diheteroaryl amino and all amino groups areprotected), —NHC(O)R (R=alkyl, cycloalkyl, aryl, aralkyl, heteroaryl orsugar), cyano; alkyl-thio-alkyl; thioalkoxy; and alkyl, cycloalkyl,aryl, alkenyl and alkynyl, which may be optionally substituted withe.g., a protected amino functionality. Preferred substitutents are2′-methoxyethyl, 2′-OCH3, 2′-O-allyl, 2′-C-allyl, and 2′-fluoro.

X³ is as described for OFG² above.

PG can be a triarylmethyl group (e.g., a dimethoxytrityl group) orSi(X^(5′))(X^(5″))(X^(5′″)) in which (X^(5′)), (X^(5″)), and (X^(5′″))are as described elsewhere.

Sugar Replacement-Based Monomers, e.g., Ligand-Conjugated Monomers(Cyclic)

Cyclic sugar replacement-based monomers, e.g., sugar replacement-basedligand-conjugated monomers, are also referred to herein as ribosereplacement monomer subunit (RRMS) monomer compounds. Preferred carriershave the general formula (LCM-2) provided below (In that structurepreferred backbone attachment points can be chosen from R¹ or R²; R³ orR⁴; or R⁹ and R¹⁰ if Y is CR⁹R¹⁰ (two positions are chosen to give twobackbone attachment points, e.g., R¹ and R⁴, or R⁴ and R⁹)). Preferredtethering attachment points include R⁷; R⁵ or R⁶ when X is CH₂. Thecarriers are described below as an entity, which can be incorporatedinto a strand. Thus, it is understood that the structures also encompassthe situations wherein one (in the case of a terminal position) or two(in the case of an internal position) of the attachment points, e.g., R¹or R²; R³ or R⁴; or R⁹ or R¹⁰ (when Y is CR⁹R¹⁰), is connected to thephosphate, or modified phosphate, e.g., sulfur containing, backbone.E.g., one of the above-named R groups can be —CH₂—, wherein one bond isconnected to the carrier and one to a backbone atom, e.g., a linkingoxygen or a central phosphorus atom.)

in which,

X is N(CO)R⁷, NR₇ or CH₂;

Y is NR⁸, O, S, CR⁹R¹⁰;

Z is CR¹¹R¹² or absent;

Each of R¹, R², R³, R⁴, R⁹, and R¹⁰ is, independently, H, OR^(a), or(CH₂)_(n)OR^(b), provided that at least two of R¹, R², R³, R⁴, R⁹, andR¹⁰ are OR^(a) and/or (CH₂)_(n)OR^(b);

Each of R⁵, R⁶, R¹¹, and R¹² is, independently, a ligand, H, C₁-C₆ alkyloptionally substituted with 1-3 R¹³, or C(O)NHR⁷; or R⁵ and R¹¹ togetherare C₃-C₈ cycloalkyl optionally substituted with R¹⁴;

R⁷ can be a ligand, e.g., R⁷ can be R^(d), or R⁷ can be a ligandtethered indirectly to the carrier, e.g., through a tethering moiety,e.g., C₁-C₂₀ alkyl substituted with NR^(c)R^(d); or C₁-C₂₀ alkylsubstituted with NHC(O)R^(d);

R⁸ is H or C₁-C₆ alkyl;

R¹³ is hydroxy, C₁-C₄ alkoxy, or halo;

R¹⁴ is NR^(c)R⁷;

R¹⁵ is C₁-C₆ alkyl optionally substituted with cyano, or C₂-C₆ alkenyl;

R¹⁶ is C₁-C₁₀ alkyl;

R¹⁷ is a liquid or solid phase support reagent;

L is —C(O)(CH₂)_(q)C(O)—, or —C(O)(CH₂)_(q)S—;

R^(a) is a protecting group, e.g., CAr₃; (e.g., a dimethoxytrityl group)or Si(X^(5′))(X^(5″))(X^(5′″)) in which (X^(5′)), (X^(5″)), and(X^(5′″)) are as described elsewhere.

R^(b) is P(O)(O⁻)H, P(OR¹⁵)N(R¹⁶)₂ or L-R¹⁷;

R^(c) is H or C₁-C₆ alkyl;

R^(d) is H or a ligand;

Each Ar is, independently, C₆-C₁₀ aryl optionally substituted with C₁-C₄alkoxy;

n is 1-4; and q is 0-4.

Exemplary carriers include those in which, e.g., X is N(CO)R⁷ or NR⁷, Yis CR⁹R¹⁰, and Z is absent; or X is N(CO)R⁷ or NR⁷, Y is CR⁹R¹⁰, and Zis CR¹¹R¹²; or X is N(CO)R⁷ or NR⁷, Y is NR⁸, and Z is CR¹¹R¹²; or X isN(CO)R⁷ or NR⁷, Y is O, and Z is CR¹¹R¹²; or X is CH₂; Y is CR⁹R¹⁰; Z isCR¹¹R¹², and R⁵ and R¹¹ together form C₆ cycloalkyl (H, z=2), or theindane ring system, e.g., X is CH₂; Y is CR⁹R¹⁰; Z is CR¹¹R¹², and R⁵and R¹¹ together form C₅ cycloalkyl (H, z=1).

In certain embodiments, the carrier may be based on the pyrroline ringsystem or the 4-hydroxyproline ring system, e.g., X is N(CO)R⁷ or NR⁷, Yis CR⁹R¹⁰, and Z is absent (D). OFG¹ is preferably attached to a primarycarbon, e.g., an exocyclic alkylene

group, e.g., a methylene group, connected to one of the carbons in thefive-membered ring (—CH₂OFG¹ in D). OFG² is preferably attached directlyto one of the carbons in the five-membered ring (—OFG² in D). For thepyrroline-based carriers, —CH₂OFG¹ may be attached to C-2 and OFG² maybe attached to C-3; or —CH₂OFG¹ may be attached to C-3 and OFG² may beattached to C-4. In certain embodiments, CH₂OFG¹ and OFG² may begeminally substituted to one of the above-referenced carbons. For the3-hydroxyproline-based carriers, —CH₂OFG¹ may be attached to C-2 andOFG² may be attached to C-4. The pyrroline- and 4-hydroxyproline-basedmonomers may therefore contain linkages (e.g., carbon-carbon bonds)wherein bond rotation is restricted about that particular linkage, e.g.restriction resulting from the presence of a ring. Thus, CH₂OFG¹ andOFG² may be cis or trans with respect to one another in any of thepairings delineated above Accordingly, all cis/trans isomers areexpressly included. The monomers may also contain one or more asymmetriccenters and thus occur as racemates and racemic mixtures, singleenantiomers, individual diastereomers and diastereomeric mixtures. Allsuch isomeric forms of the monomers are expressly included (e.g., thecenters bearing CH₂OFG¹ and OFG² can both have the R configuration; orboth have the S configuration; or one center can have the Rconfiguration and the other center can have the S configuration and viceversa). The tethering attachment point is preferably nitrogen. Preferredexamples of carrier D include the following:

In certain embodiments, the carrier may be based on the piperidine ringsystem (E), e.g., X is N(CO)R⁷ or NR⁷, Y is CR⁹R¹⁰, and Z is CR¹¹R¹².OFG¹ is preferably

attached to a primary carbon, e.g., an exocyclic alkylene group, e.g., amethylene group (n=1) or ethylene group (n=2), connected to one of thecarbons in the six-membered ring [—(CH₂)_(n)OFG¹ in E]. OFG² ispreferably attached directly to one of the carbons in the six-memberedring (—OFG² in E). —(CH₂)_(n)OFG¹ and OFG² may be disposed in a geminalmanner on the ring, i.e., both groups may be attached to the samecarbon, e.g., at C-2, C-3, or C-4. Alternatively, —(CH₂)_(n)OFG¹ andOFG² may be disposed in a vicinal manner on the ring, i.e., both groupsmay be attached to adjacent ring carbon atoms, e.g.,

—(CH₂)_(n)OFG¹ may be attached to C-2 and OFG² may be attached to C-3;—(CH₂)_(n)OFG¹ may be attached to C-3 and OFG² may be attached to C-2;—(CH₂)_(n)OFG¹ may be attached to C-3 and OFG² may be attached to C-4;or —(CH₂)_(n)OFG¹ may be attached to C-4 and OFG² may be attached toC-3. The piperidine-based monomers may therefore contain linkages (e.g.,carbon-carbon bonds) wherein bond rotation is restricted about thatparticular linkage, e.g. restriction resulting from the presence of aring. Thus, —(CH₂)_(n)OFG¹ and OFG² may be cis or trans with respect toone another in any of the pairings delineated above. Accordingly, allcis/trans isomers are expressly included. The monomers may also containone or more asymmetric centers and thus occur as racemates and racemicmixtures, single enantiomers, individual diastereomers anddiastereomeric mixtures. All such isomeric forms of the monomers areexpressly included (e.g., the centers bearing CH₂OFG¹ and OFG² can bothhave the R configuration; or both have the S configuration; or onecenter can have the R configuration and the other center can have the Sconfiguration and vice versa). The tethering attachment point ispreferably nitrogen.

In certain embodiments, the carrier may be based on the piperazine ringsystem (F), e.g., X is N(CO)R⁷ or NR⁷, Y is NR⁸, and Z is CR¹¹R¹², orthe morpholine ring system (G), e.g., X is N(CO)R⁷ or NR⁷, Y is O, and Zis CR¹¹R¹². OFG¹ is preferably

attached to a primary carbon, e.g., an exocyclic alkylene group, e.g., amethylene group, connected to one of the carbons in the six-memberedring (—CH₂OFG¹ in F or G). OFG² is preferably attached directly to oneof the carbons in the six-membered rings (—OFG² in F or G). For both Fand G, —CH₂OFG¹ may be attached to C-2 and OFG² may be attached to C-3;or vice versa. In certain embodiments, CH₂OFG¹ and OFG² may be geminallysubstituted to one of the above-referenced carbons. The piperazine- andmorpholine-based monomers may therefore contain linkages (e.g.,carbon-carbon bonds) wherein bond rotation is restricted about thatparticular linkage, e.g. restriction resulting from the presence of aring. Thus, CH₂OFG¹ and OFG² may be cis or trans with respect to oneanother in any of the pairings delineated above. Accordingly, allcis/trans isomers are expressly included. The monomers may also containone or more asymmetric centers and thus occur as racemates and racemicmixtures, single enantiomers, individual diastereomers anddiastereomeric mixtures. All such isomeric forms of the monomers areexpressly included (e.g., the centers bearing CH₂OFG¹ and OFG² can bothhave the R configuration; or both have the S configuration; or onecenter can have the R configuration and the other center can have the Sconfiguration and vice versa). R′″ can be, e.g., C₁-C₆ alkyl, preferablyCH₃. The tethering attachment point is preferably nitrogen in both F andG.

In certain embodiments, the carrier may be based on the decalin ringsystem, e.g., X is CH₂; Y is CR⁹R¹⁰; Z is CR¹¹R¹², and R⁵ and R¹¹together form C₆ cycloalkyl (H, z=2), or the indane ring system, e.g., Xis CH₂; Y is CR⁹R¹⁰; Z is CR¹¹R¹², and R⁵ and R¹¹ together form C₅cycloalkyl (H, z=1). OFG¹ is preferably attached to a primary carbon,

e.g., an exocyclic methylene group (n=1) or ethylene group (n=2)connected to one of C-2, C-3, C-4, or C-5 [—(CH₂)_(n)OFG¹ in H]. OFG² ispreferably attached directly to one of C-2, C-3, C-4, or C-5 (—OFG² inH). —(CH₂)_(n)OFG¹ and OFG² may be disposed in a geminal manner on thering, i.e., both groups may be attached to the same carbon, e.g., atC-2, C-3, C-4, or C-5. Alternatively, —(CH₂)_(n)OFG¹ and OFG² may bedisposed in a vicinal manner on the ring, i.e., both groups may beattached to adjacent ring carbon atoms, e.g., —(CH₂)_(n)OFG¹ may beattached to C-2 and OFG² may be attached to C-3; —(CH₂)_(n)OFG¹ may beattached to C-3 and OFG² may be attached to C-2; —(CH₂)_(n)OFG¹ may beattached to C-3 and OFG² may be attached to C-4; or —(CH₂)_(n)OFG¹ maybe attached to C-4 and OFG² may be attached to C-3; —(CH₂)_(n)OFG¹ maybe attached to C-4 and OFG² may be attached to C-5; or —(CH₂)_(n)OFG¹may be attached to C-5 and OFG² may be attached to C-4. The decalin orindane-based monomers may therefore contain linkages (e.g.,carbon-carbon bonds) wherein bond rotation is restricted about thatparticular linkage, e.g. restriction resulting from the presence of aring. Thus, —(CH₂)_(n)OFG¹ and OFG² may be cis or trans with respect toone another in any of the pairings delineated above. Accordingly, allcis/trans isomers are expressly included. The monomers may also containone or more asymmetric centers and thus occur as racemates and racemicmixtures, single enantiomers, individual diastereomers anddiastereomeric mixtures. All such isomeric forms of the monomers areexpressly included (e.g., the centers bearing CH₂OFG¹ and OFG² can bothhave the R configuration; or both have the S configuration; or onecenter can have the R configuration and the other center can have the Sconfiguration and vice versa). In a preferred embodiment, thesubstituents at C-1 and C-6 are trans with respect to one another. Thetethering attachment point is preferably C-6 or C-7.

Other carriers may include those based on 3-hydroxyproline (J). Thus,—(CH₂)_(n)OFG¹ and OFG² may be cis or trans with respect to one another.Accordingly, all cis/trans isomers are expressly included. The monomersmay also contain one or more asymmetric centers

and thus occur as racemates and racemic mixtures, single enantiomers,individual diastereomers and diastereomeric mixtures. All such isomericforms of the monomers are expressly included (e.g., the centers bearingCH₂OFG¹ and OFG² can both have the R configuration; or both have the Sconfiguration; or one center can have the R configuration and the othercenter can have the S configuration and vice versa). The tetheringattachment point is preferably nitrogen.

Representative cyclic, sugar replacement-based carriers are shown inFIG. 3.

Sugar Replacement-Based Monomers (Acyclic)

Acyclic sugar replacement-based monomers, e.g., sugar replacement-basedligand-conjugated monomers, are also referred to herein as ribosereplacement monomer subunit (RRMS) monomer compounds. Preferred acycliccarriers can have formula LCM-3 or LCM-4 below.

In some embodiments, each of x, y, and z can be, independently of oneanother, 0, 1, 2, or 3. In formula LCM-3, when y and z are different,then the tertiary carbon can have either the R or S configuration. Inpreferred embodiments, x is zero and y and z are each 1 in formula LCM-3(e.g., based on serinol), and y and z are each 1 in formula LCM-3. Eachof formula LCM-3 or LCM-4 below can optionally be substituted, e.g.,with hydroxy, alkoxy, perhaloalkyl.

Tethers

In some embodiments, a moiety, e.g., a ligand may be connectedindirectly to the carrier via the intermediacy of an intervening tether.Tethers are connected to the carrier at a tethering attachment point(TAP).

Tethers can include any C1-C100 carbon-containing moiety, (e.g. C1-C75,C1-C50, C1-C20, C1-C10; C1, C2, C3, C4, C5, C6, C7, C8, C9, or C10) andat least one linking group (e.g. at least two linking groups, at leastthree linking groups, at least four linking groups, at least fivelinking groups, at least six linking groups, at least seven linkinggroups, at least eight linking groups, at least nine linking groups, atleast ten linking groups, at least eleven linking groups, at leasttwelve linking groups, at least thirteen linking groups, at leastfourteen linking groups, at least fifteen linking groups, at leastsixteen linking groups, at least seventeen linking groups, at leasteighteen linking groups, at least nineteen linking groups, at leasttwenty linking groups). The linking group can be at one or both terminalpositions of the tether (e.g., a terminal linking group can link thetether to the ligand or link the tether to the nitrogen atom of X or R¹⁴in formula (I), or the nitrogen atom of CONHR⁷ when R⁵, R⁶, R¹¹, or R¹²is CONHR⁷ in formula (I)) and/or at one or more internal positions ofthe tether (1 carbon from the end, 2 carbons from the end, 3 carbonsfrom the end, 4 carbons from the end, 5 carbons from the end, 3 carbonsfrom the end, etc.).

In general, the linking group can be any atom or group of atoms thatincludes as a linking atom a divalent, trivalent, tetravalent,pentavalent or hexavalent heteroatom, e.g. O, N, S, P. A carbon atom canbe the linking atom of a linking group provided that the linking carbonatom is doubly bonded to a non-linking heteroatom (e.g., carbonyl,—C(O)—). Linking groups can include without limitation amides, ethers,esters, phosphates, carbamates, thioethers, disulphide, thioamide,thioester, thiocarbamate, thiophosphate, carbonyl, amino, and hydrazone.Exemplary linking groups could include without limitation —NR^(k)C(O)—,—C(O)NR^(k)—, —OC(O)NR^(k)—, —NR^(k)C(O)O—, —O—, —S—, —SS—, —S(O)—,—S(O₂)—, —NR^(k)C(O)NR^(k)—, —NR^(k)C(S)NR^(k)—, —C(O)O—, —OC(O)—,—NR^(k)C(S)—, —NR^(k)C(S)O—, —C(S)NR^(k)—, —OC(S)NR^(k)—, —NR^(k)C(S)O—,—O—P(O)(OR^(k))—O—, —O—P(S)(OR^(k))—O—, —O—P(S)(SR^(k))—O—,—S—P(O)(OR^(k))—O—, —O—P(O)(OR^(k))—S—, —S—P(O)(OR^(k))—S—,—O—P(S)(OR^(k))—S—, —S—P(S)(OR^(k))—O—, —O—P(O)(R^(k))—O—,—O—P(S)(R^(k))—O—, —S—P(O)(R^(k))—O—, —S—P(S)(R^(k))—O—,—S—P(O)(R^(k))—S—, —O—P(S)(R^(k))—S—, —C(O)—, —NR^(k)— —R^(k)C═NNR^(k)—,P1′C(O)NHP2′-, or —P1′NHC(O)P2′.

In general, at least one of the linking groups in the tether is acleavable linking group. A cleavable linking group is one which issufficiently stable outside the cell such that it allows targeting of atherapeutically beneficial amount of an iRNA agent (e.g., a singlestranded or double stranded iRNA agent), coupled by way of the cleavablelinking group to a targeting agent—to targets cells, but which uponentry into a target cell is cleaved to release the iRNA agent from thetargeting agent. In a preferred embodiment, the cleavable linking groupis cleaved at least 10 times or more, preferably at least 100 timesfaster in the target cell or under a first reference condition (whichcan, e.g., be selected to mimic or represent intracellular conditions)than in the blood of a subject, or under a second reference condition(which can, e.g., be selected to mimic or represent conditions found inthe blood or serum).

Cleavable Linking Groups

Cleavable linking groups are susceptible to cleavage agents, e.g., pH,redox potential or the presence of degradative molecules. Generally,cleavage agents are more prevalent or found at higher levels oractivities inside cells than in serum or blood. Examples of suchdegradative agents include: redox agents which are selected forparticular substrates or which have no substrate specificity, including,e.g., oxidative or reductive enzymes or reductive agents such asmercaptans, present in cells, that can degrade a redox cleavable linkinggroup by reduction; esterases; endosomes or agents that can create anacidic environment, e.g., those that result in a pH of five or lower;enzymes that can hydrolyze or degrade an acid cleavable linking group byacting as a general acid, peptidases (which can be substrate specific),and phosphatases.

A cleavable linkage group, such as a disulfide bond can be susceptibleto pH. The pH of human serum is 7.4, while the average intracellular pHis slightly lower, ranging from about 7.1-7.3. Endosomes have a moreacidic pH, in the range of 5.5-6.0, and lysosomes have an even moreacidic pH at around 5.0. Some tethers will have a linkage group that iscleaved at a preferred pH, thereby releasing the iRNA agent from aligand (e.g., a targeting or cell-permeable ligand, such as cholesterol)inside the cell, or into the desired compartment of the cell.

A chemical junction (e.g., a linking group) that links a ligand to aniRNA agent can include a disulfide bond. When the iRNA agent/ligandcomplex is taken up into the cell by endocytosis, the acidic environmentof the endosome will cause the disulfide bond to be cleaved, therebyreleasing the iRNA agent from the ligand (Quintana et al., Pharm Res.19:1310-1316, 2002; Patri et al., Curr. Opin. Curr. Biol. 6:466-471,2002). The ligand can be a targeting ligand or a second therapeuticagent that may complement the therapeutic effects of the iRNA agent.

A tether can include a linking group that is cleavable by a particularenzyme. The type of linking group incorporated into a tether can dependon the cell to be targeted by the iRNA agent. For example, an iRNA agentthat targets an mRNA in liver cells can be conjugated to a tether thatincludes an ester group. Liver cells are rich in esterases, andtherefore the tether will be cleaved more efficiently in liver cellsthan in cell types that are not esterase-rich. Cleavage of the tetherreleases the iRNA agent from a ligand that is attached to the distal endof the tether, thereby potentially enhancing silencing activity of theiRNA agent. Other cell-types rich in esterases include cells of thelung, renal cortex, and testis.

Tethers that contain peptide bonds can be conjugated to iRNA agentstarget to cell types rich in peptidases, such as liver cells andsynoviocytes. For example, an iRNA agent targeted to synoviocytes, suchas for the treatment of an inflammatory disease (e.g., rheumatoidarthritis), can be conjugated to a tether containing a peptide bond.

In general, the suitability of a candidate cleavable linking group canbe evaluated by testing the ability of a degradative agent (orcondition) to cleave the candidate linking group. It will also bedesirable to also test the candidate cleavable linking group for theability to resist cleavage in the blood or when in contact with othernon-target tissue, e.g., tissue the iRNA agent would be exposed to whenadministered to a subject. Thus one can determine the relativesusceptibility to cleavage between a first and a second condition, wherethe first is selected to be indicative of cleavage in a target cell andthe second is selected to be indicative of cleavage in other tissues orbiological fluids, e.g., blood or serum. The evaluations can be carriedout in cell free systems, in cells, in cell culture, in organ or tissueculture, or in whole animals. It may be useful to make initialevaluations in cell-free or culture conditions and to confirm by furtherevaluations in whole animals. In preferred embodiments, useful candidatecompounds are cleaved at least 2, 4, 10 or 100 times faster in the cell(or under in vitro conditions selected to mimic intracellularconditions) as compared to blood or serum (or under in vitro conditionsselected to mimic extracellular conditions).

Redox Cleavable Linking Groups

One class of cleavable linking groups are redox cleavable linking groupsthat are cleaved upon reduction or oxidation. An example of reductivelycleavable linking group is a disulphide linking group (—S—S—). Todetermine if a candidate cleavable linking group is a suitable“reductively cleavable linking group,” or for example is suitable foruse with a particular iRNA moiety and particular targeting agent one canlook to methods described herein. For example, a candidate can beevaluated by incubation with dithiothreitol (DTT), or other reducingagent using reagents know in the art, which mimic the rate of cleavagewhich would be observed in a cell, e.g., a target cell. The candidatescan also be evaluated under conditions which are selected to mimic bloodor serum conditions. In a preferred embodiment, candidate compounds arecleaved by at most 10% in the blood. In preferred embodiments, usefulcandidate compounds are degraded at least 2, 4, 10 or 100 times fasterin the cell (or under in vitro conditions selected to mimicintracellular conditions) as compared to blood (or under in vitroconditions selected to mimic extracellular conditions). The rate ofcleavage of candidate compounds can be determined using standard enzymekinetics assays under conditions chosen to mimic intracellular media andcompared to conditions chosen to mimic extracellular media.

Phosphate-Based Cleavable Linking Groups

Phosphate-based linking groups are cleaved by agents that degrade orhydrolyze the phosphate group. An example of an agent that cleavesphosphate groups in cells are enzymes such as phosphatases in cells.Examples of phosphate-based linking groups are —O—P(O)(ORk)-O—,—O—P(S)(ORk)-O—, —O—P(S)(SRk)-O—, —S—P(O)(ORk)-O—, —O—P(O)(ORk)-S—,—S—P(O)(ORk)-S—, —O—P(S)(ORk)-S—, —S—P(S)(ORk)-O—, —O—P(O)(Rk)-O—,—O—P(S)(Rk)-O—, —S—P(O)(Rk)-O—, —S—P(S)(R))-—, —S—P(O)(Rk)-S—,—O—P(S)(R^(k))—S—. Preferred embodiments are —O—P(O)(OH)—O—,—O—P(S)(OH)—O—, —O—P(S)(SH)—O—, —S—P(O)(OH)—O—, —O—P(O)(OH)—S—,—S—P(O)(OH)—S—, —O—P(S)(OH)—S—, —S—P(S)(OH)—O—, —O—P(O)(H)—O—,—O—P(S)(H)—O—, —S—P(O)(H)—O—, —S—P(S)(H)—O—, —S—P(O)(H)—S—,—O—P(S)(H)—S—. A preferred embodiment is —O—P(O)(OH)—O—. Thesecandidates can be evaluated using methods analogous to those describedabove.

Acid Cleavable Linking Groups

Acid cleavable linking groups are linking groups that are cleaved underacidic conditions. In preferred embodiments acid cleavable linkinggroups are cleaved in an acidic environment with a pH of about 6.5 orlower (e.g., about 6.0, 5.5, 5.0, or lower), or by agents such asenzymes that can act as a general acid. In a cell, specific low pHorganelles, such as endosomes and lysosomes can provide a cleavingenvironment for acid cleavable linking groups. Examples of acidcleavable linking groups include but are not limited to hydrazones,esters, and esters of amino acids. Acid cleavable groups can have thegeneral formula —C═NN—, C(O)O, or —OC(O). A preferred embodiment is whenthe carbon attached to the oxygen of the ester (the alkoxy group) is anaryl group, substituted alkyl group, or tertiary alkyl group such asdimethyl pentyl or t-butyl. These candidates can be evaluated usingmethods analogous to those described above.

Ester-Based Linking Groups

Ester-based linking groups are cleaved by enzymes such as esterases andamidases in cells. Examples of ester-based cleavable linking groupsinclude but are not limited to esters of alkylene, alkenylene andalkynylene groups. Ester cleavable linking groups have the generalformula —C(O)O—, or —OC(O)—. These candidates can be evaluated usingmethods analogous to those described above.

Peptide-Based Cleaving Groups

Peptide-based linking groups are cleaved by enzymes such as peptidasesand proteases in cells. Peptide-based cleavable linking groups arepeptide bonds formed between amino acids to yield oligopeptides (e.g.,dipeptides, tripeptides etc.) and polypeptides. Peptide-based cleavablegroups do not include the amide group (—C(O)NH—). The amide group can beformed between any alkylene, alkenylene or alkynelene. A peptide bond isa special type of amide bond formed between amino acids to yieldpeptides and proteins. The peptide based cleavage group is generallylimited to the peptide bond (i.e., the amide bond) formed between aminoacids yielding peptides and proteins and does not include the entireamide functional group. Peptide cleavable linking groups have thegeneral formula —NHCHR¹C(O)NHCHR²C(O)—, where R¹ and R² are the R groupsof the two adjacent amino acids. These candidates can be evaluated usingmethods analogous to those described above.

The carbon containing moiety (e.g., hydrocarbon moiety, partial or fullyhalocarbon moiety) can be cyclic or acyclic moiety or any combinationthereof. In some embodiments, the carbon containing moiety can besaturated (e.g., alkylene) or unsaturated (e.g., alkenylene oralkynylene).

In certain embodiments, the saturated carbon containing moiety can beC1-C100 (e.g. C1-C75, C1-C50, C1-C20, C1-C10; C1, C2, C3, C4, C5, C6,C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, or C20)alkylene moiety, which may be straight chain or branched. In certainembodiments, the saturated carbon can be further substituted with halo,hydroxy or amino.

In certain embodiments, the unsaturated carbon containing moiety can beC1-C100 (e.g. C1-C75, C1-C50, C1-C20, C1-C10; C1, C2, C3, C4, C5, C6,C7, C8, C9, C₁₀, C11, C12, C13, C14, C15, C16, C17, C18, C19, or C20)alkenylene moiety, which may be straight chain or branched. In certainembodiments, the alkenylene moieties can have one or more double bonds.In certain embodiments, the alkenylene moieties can have 1 to 60 doublebonds (e.g., 1-50. 1-40, 1-30, 1-20, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, or 20 double bonds). In general, eachdouble bond can be independently of one another cis or trans or E or Z,or any combination thereof. In certain embodiments, the unsaturatedcarbon moiety can be (CH₂)_(m1)(CH═CH)_(m2)(CH₂)m3, in which each of m1,m2, or m3 is, independently of one another, 0-20 (e.g., 0, 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20).

In certain embodiments, the unsaturated carbon containing moiety can beC1-C100 (e.g. C1-C75, C1-C50, C1-C20, C1-C10; C1, C2, C3, C4, C5, C6,C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, or C20)alkynylene moiety. In certain embodiments, the alkynylene moieties canhave one or more triple bonds. In certain embodiments, the alkynylenemoieties can have one to sixty triple bonds (e.g., 1-50. 1-40, 1-30,1-20, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,or 20 triple bonds). In certain embodiments, the carbon containingmoiety can be substituted or unsubstituted. In certain embodiments, thecarbon containing moiety can be straight or branched.

In certain embodiments, the unsaturated carbon containing moieties canbe substituted or unsubstituted. In certain embodiments, the unsaturatedcarbon containing moiety can be straight or branched.

In certain embodiments, the carbon containing moiety can be cyclic orinclude a series of linked cyclic moieties. In certain embodiments, thecyclic moieties can be cycloalkyl groups. In certain embodiments, thecyclic moieties can be cycloalkenyl groups. In certain embodiments, thecyclic moieties can be cycloalkynyl groups. In certain embodiments, thecyclic moieties can be heterocyclyl groups. In certain embodiments, thecyclic moieties can be aryl groups. In certain embodiments, the cyclicmoieties can be heteroaryl groups. In certain embodiments, the cyclicmoieties can be phenyl, pyridinyl, pyranyl, or thiophenyl groups. Incertain embodiments, the cyclic moieties can be carbohydrate or sugargroups. In certain embodiments, the carbohydrate moieties can beglucose, mannose or ribose groups.

In certain embodiments, the tether can have the formula shown below.T=-(E′)_(s)-Δ-(E″)_(t)-Formula T₁′,

In which, E′ is a terminal linking group that is linking the tether tothe ligand. E″ is a tether linking group that links tether to thetethering attachment point (TAP); Δ is a hydrocarbon chain e.g.alkylene, alkenylene, or alkynylene that optionally has one or moreinternal linking groups G; and s and t can be 0 or 1. In allembodiments, one of s and t is one, or Δ includes at least one G, and atleast one of the linking groups is a cleavable linking group.

When present, there terminal linking groups E′ and E″ can be selected asdesired.

In certain embodiments, one or both of the terminal linking groups E′and E″ can be cleavable linking groups (e.g., —SS—, —O—P(O)(OR^(k))—O—,—O—P(S)(OR^(k))—O—, —O—P(S)(SR^(k))—O—, —S—P(O)(OR^(k))—O—,—O—P(O)(OR^(k))—S—, —S—P(O)(OR^(k))—S—, —O—P(S)(OR^(k))—S—,—S—P(S)(OR^(k))—O—, —O—P(O)(R^(k))—O—, —O—P(S)(R^(k))—O—,—S—P(O)(R^(k))—O—, —S—P(S)(R^(k))—O—, —S—P(O)(R^(k))—S—,—O—P(S)(R^(k))—S—, —C(O)O—, —OC(O)—, —R^(k)C═NNR^(k)—,NHCHR^(k′)C(O)NHCHR^(k″)C(O)—, or —NHCHR^(k′)NHC(O)CHR^(k″)C(O)—).

In some embodiments, s can be 0 and t can be 1, i.e. E″ is present andE′ is absent; and E″ can be NR^(k)C(O)—, —C(O)NR^(k)—, —OC(O)NR^(k)—,—NR^(k)C(O)O—, —O—, —S—, —SS—, —S(O)—, —S(O₂)—, —NR^(k)C(O)NR^(k)—,—NR^(k)C(S)NR^(k)—, —C(O)O—, —OC(O)—, —NR^(k)C(S)—, —NR^(k)C(S)O—,—C(S)NR^(k)—, —OC(S)NR^(k)—, —NR^(k)C(S)O—, —O—P(O)(OR^(k))—O—,—O—P(S)(OR^(k))—O—, —O—P(S)(SR^(k))—O—, —S—P(O)(OR^(k))—O—,—O—P(O)(OR^(k))—S—, —S—P(O)(OR^(k))—S—, —O—P(S)(OR^(k))—S—,—S—P(S)(OR^(k))—O—, —O—P(O)(R^(k))—O—, —O—P(S)(R^(k))—O—,—S—P(O)(R^(k))—O—, —S—P(S)(R^(k))—O—, —S—P(O)(R^(k))—S—,—O—P(S)(R^(k))—S—, —C(O)—, —NR^(k)— —R^(k)C═NNR^(k)—,NHCHR^(k′)C(O)NHCHR^(k″)C(O)—, or —NHCHR^(k′)NHC(O)CHR^(k″)C(O)—. Inpreferred embodiments, E″ is C(O)NRk-, —NHC(O)—, —NHC(O)O—, OC(O)NRk- or—NHC(O)NRk-.

In some embodiments, s can be 1 and t can be 0, i.e., E′ is present andE″ is absent, and E′ can be NR^(k)C(O)—, —C(O)NR^(k)—, —OC(O)NR^(k)—,—NR^(k)C(O)O—, —O—, —S—, —SS—, —S(O)—, —S(O₂)—, —NR^(k)C(O)NR^(k)—,—NR^(k)C(S)NR^(k)—, —C(O)O—, —OC(O)—, —NR^(k)C(S)—, —NR^(k)C(S)O—,—C(S)NR^(k)—, —OC(S)NR^(k)—, —NR^(k)C(S)O—, —O—P(O)(OR^(k))—O—,—O—P(S)(OR^(k))—O—, —O—P(S)(SR^(k))—O—, —S—P(O)(OR^(k))—O—,—O—P(O)(OR^(k))—S—, —S—P(O)(OR^(k))—S—, —O—P(S)(OR^(k))—S—,—S—P(S)(OR^(k))—O—, —O—P(O)(R^(k))—O—, —O—P(S)(R^(k))—O—,—S—P(O)(R^(k))—O—, —S—P(S)(R^(k))—O—, —S—P(O)(R^(k))—S—,—O—P(S)(R^(k))—S—, —C(O)—, —NR^(k)— —R^(k)C═NNR^(k)—,NHCHR^(k′)C(O)NHCHR^(k″)C(O)—, or —NHCHR^(k′)NHC(O)CHR^(k″)C(O)—. Inpreferred embodiments, E′ can be C(O)NH—, —NH—, OC(O)NRk- or —SS—.

In some embodiments, s and t can both be 1. In certain embodiments, oneof E′ and E″ can be carbamates (—NHC(O)O—), ethers (—O—), amides(—C(O)NH—), thioethers (—S—), thioamide (—C(S)NH—), oxo (—C(O)—), amino(—NH—), and the other of E′ and E″ can be esters (—C(O)O—, —OC(O)—),phosphates (—O—P(O)(ORk)-O—, —O—P(S)(ORk)-O—, —O—P(S)(SRk)-O—,—S—P(O)(ORk)-O—, —O—P(O)(ORk)-S—, —S—P(O)(ORk)-S—, —O—P(S)(ORk)-S—,—S—P(S)(ORk)-O—, —O—P(O)(Rk)-O—, —O—P(S)(Rk)-O—, —S—P(O)(Rk)-O—,—S—P(S)(Rk)-O—, —S—P(O)(Rk)-S—, —O—P(S)(Rk)-S—), disu lphide (—SS—),hydrazone (R^(k)C═NNR^(k)—), and peptides P1′C(O)NHP2′-, or—P1′NHC(O)P2′.

In a certain embodiment, one of E′ and E″ can be —C(O)NRk-, —NHC(O)—,—NHC(O)O—, —OC(O)NH— or —SS— and the other of E′ and E″ can be—C(O)NRk-, —NHC(O)—, —NHC(O)O—, or —NHC(O)NRk-.

Δ can be any hydrocarbon chain and can optionally have one or moreinternal groups, G. In some embodiments, Δ includes at least oneinternal linking group G.

As used herein, the term Δ=C4 alkylene having 1 G (and the like) caninclude for example and without limitation the moieties delineated informulae T-2 and T-3:—CH₂—CH₂—S—S—CH₂—CH₂—  T-2—CH₂—CH₂—C(O)—CH₂—CH₂—,  T-3

in which G is —S—S— and —C(O)— in formulae T-2 and T-3 respectively.

In certain embodiments, Δ can be C1-C20 (e.g. C1, C2, C3, C4, C5, C6,C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, or C20)alkylene, alkenylene, or alkynylene and include 0 to 10 (e.g., 0, 1, 2,3, 4, 5, 6, 7, 8, 9, or 10) G groups.

In certain embodiments, Δ can be C2-C20 (C2, C3, C4, C5, C6, C7, C8, C9,C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, or C20) alkylene,having 1 to 10 (1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) G groups.

In certain embodiments, Δ can be C2-C20 (C2, C3, C4, C5, C6, C7, C8, C9,C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, or C20) alkenylene,having 1 to 10 (1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) G groups.

In certain embodiments, Δ can be C2-C20 (C2, C3, C4, C5, C6, C7, C8, C9,C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, or C20) alkynylene,having 1 to 10 (1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) G groups.

In certain embodiments, Δ can be C2-C20 (e.g. C1, C2, C3, C4, C5, C6,C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, or C20)alkylene, alkenylene, or alkynylene and include 1 to 5 (e.g., 1, 2, 3,4, or 5) G groups.

In certain embodiments, Δ can be C2-C20 (C2, C3, C4, C5, C6, C7, C8, C9,C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, or C20) alkylenehaving 0-10 (0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) G groups. In certainembodiments, Δ can be C16 alkylene having 1-4 (1, 2, 3, or 4) G groups,preferably 3 G groups. In certain embodiments, Δ can be C14 alkylenehaving 1-4 (1, 2, 3, or 4) G groups, preferably 3 G groups. In certainembodiments, Δ can be C12 alkylene having 1-4 (1, 2, 3, or 4) G groups,preferably 3 G groups. In certain embodiments, Δ can be C9 alkylenehaving 1-3 (1, 2, or 3) G groups, preferably 3 G groups. In certainembodiments, Δ can be C6 alkylene having 1-4 (1, 2, 3, or 4) G groups,preferably 3 G groups.

When present G can be selected as desired from e.g. NR^(k)C(O)—,—C(O)NR^(k)—, —OC(O)NR^(k)—, NR^(k)C(O)O—, —O—, —S—, —SS—, —S(O)—,—S(O₂)—, —NR^(k)C(O)NR^(k)—, —NR^(k)C(S)NR^(k)—, —C(O)O—, —OC(O)—,—NR^(k)C(S)—, —NR^(k)C(S)O—, —C(S)NR^(k)—, —OC(S)NR^(k)—, —NR^(k)C(S)O—,—O—P(O)(OR^(k))—O—, —O—P(S)(OR^(k))—O—, —O—P(S)(SR^(k))—O—,—S—P(O)(OR^(k))—O—, —O—P(O)(OR^(k))—S—, —S—P(O)(OR^(k))—S—,—O—P(S)(OR^(k))—S—, —S—P(S)(OR^(k))—O—, —O—P(O)(R^(k))—O—,—O—P(S)(R^(k))—O—, —S—P(O)(R^(k))—O—, —S—P(S)(R^(k))—O—,—S—P(O)(R^(k))—S—, —O—P(S)(R^(k))—S—, —C(O)—, —NR^(k)— —R^(k)C═NNR^(k)—,NHCHR^(k′)C(O)NHCHR^(k″)C(O)—, or —NHCHR^(k′)NHC(O)CHR^(k″)C(O)—.

In some embodiments, G can be a cleavable internal linking group (e.g.,disulphide, ester, hydrazone, peptide or phosphate, e.g., —SS—,—O—P(O)(OR^(k))—O—, —O—P(S)(OR^(k))—O—, —O—P(S)(SR^(k))—O—,—S—P(O)(OR^(k))—O—, —O—P(O)(OR^(k))—S—, —S—P(O)(OR^(k))—S—,—O—P(S)(OR^(k))—S—, —S—P(S)(OR^(k))—O—, —O—P(O)(R^(k))—O—,—O—P(S)(R^(k))—O—, —S—P(O)(R^(k))—O—, —S—P(S)(R^(k))—O—,—S—P(O)(R^(k))—S—, —O—P(S)(R^(k))—S—, —C(O)O—, —OC(O)—,—R^(k)C═NNR^(k)—, NHCHR^(k′)C(O)NHCHR^(k″)C(O)—, or—NHCHR^(k′)NHC(O)CHR^(k″)C(O)—).

In certain embodiments two or more (2, 3, 4, or 5) G groups can bepresent, and one G can be carbamates (—NHC(O)O—), ethers (—O—), amides(—C(O)NH—), thioethers (—S—), thioamide (—C(S)NH—), oxo (—C(O)—), amino(—NH—), and the remaining G groups can be esters (—C(O)O—, —OC(O)—),phosphates (—O—P(O)(ORk)-O—, —O—P(S)(ORk)-O—, —O—P(S)(SRk)-O—,—S—P(O)(ORk)-O—, —O—P(O)(ORk)-S—, —S—P(O)(ORk)-S—, —O—P(S)(ORk)-S—,—S—P(S)(ORk)-O—, —O—P(O)(Rk)-O—, —O—P(S)(Rk)-O—, —S—P(O)(Rk)-O—,—S—P(S)(Rk)-O—, —S—P(O)(Rk)-S—, —O—P(S)(Rk)-S—), disulphide (—SS—),hydrazone (R^(k)C═NNR^(k)—), and peptides NHCHR^(k′)C(O)NHCHR^(k″)C(O)—,or —NHCHR^(k′)NHC(O)CHR^(k″)C(O)—. Preferred G groups include —C(O)NH—,—SS—, —NHC(O)NH—, —C(O)N—, NHC(O)N, —NHC(O)—.

In certain embodiments, G can be an ether, amino, or thioether linkinggroups, and Δ can include a polyether or polyimino moiety (e.g.,(CH2CH2O)m1(CH2)m2) or (CH2CH2NH)m1(CH2)m2) in which m1 and m2 can be0-20.

In some embodiments, Δ can have the formula T-4 shown below:T-4=-(Q¹)f ₁-(G¹)j ₁-(Q²)f ₂-(G²)j ₂-(Q³)f ₃-(G³)j ₃-(Q⁴)f ₄-(G⁴)j₄-(Q⁵)f ₅

in which each of Q1, Q2, Q3, Q4, and Q5 independently of one another canbe C1-C10, e.g. (C1, C2, C3, C4, C5, C6, C7, C8, C9, or C10) alkyleneeach of G1, G2, G3, and G4 can be, e.g., —SS—, —O—P(O)(OR^(k))—O—,—O—P(S)(OR^(k))—O—, —O—P(S)(SR^(k))—O—, —S—P(O)(OR^(k))—O—,—O—P(O)(OR^(k))—S—, —S—P(O)(OR^(k))—S—, —O—P(S)(OR^(k))—S—,—S—P(S)(OR^(k))—O—, —O—P(O)(R^(k))—O—, —O—P(S)(R^(k))—O—,—S—P(O)(R^(k))—O—, —S—P(S)(R^(k))—O—, —S—P(O)(R^(k))—S—,—O—P(S)(R^(k))—S—, —C(O)O—, —OC(O)—, —R^(k)C═NNR^(k)—, P1′C(O)NHP2′-,—P1′NHC(O)P2′, NR^(k)C(O)—, —C(O)NR^(k)—, —OC(O)NR^(k)—, —NR^(k)C(O)O—,—O—, —S—, NR^(k)C(O)NR^(k)—, —NR^(k)C(S)NR^(k)—, —NR^(k)C(S)—,—NR^(k)—C(S)O—, —C(S)NR^(k)—, —OC(S)NR^(k)—, —NR^(k)C(S)O, —C(O)—, or—NR^(k)—, where f and j can be 0 or 1.

In certain embodiments, f₁, f₂, j₁ can be 1 and, f₃, f₄, f₅, j₂, j₃, andj₄ can be 0, i.e., Q1-G1-Q2 are present. In certain embodiments each ofQ1 and Q2 can be C1 to C5 (e.g. C1, C2, C3, C4, C5) alkylene, G1 can beselected from —SS—, —O—P(O)(OR^(k))—O—, —O—P(S)(OR^(k))—O—,—O—P(S)(SR^(k))—O—, —S—P(O)(OR^(k))—O—, —O—P(O)(OR^(k))—S—,—S—P(O)(OR^(k))—S—, —O—P(S)(OR^(k))—S—, —S—P(S)(OR^(k))—O—,—O—P(O)(R^(k))—O—, —O—P(S)(R^(k))—O—, —S—P(O)(R^(k))—O—,—S—P(S)(R^(k))—O—, —S—P(O)(R^(k))—S—, —O—P(S)(R^(k))—S—, —C(O)O—,—OC(O)—, —R^(k)C═NNR^(k)—, P1′C(O)NHP2′-, or —P1′NHC(O)P2′. In apreferred embodiment, each of Q1 and Q2 is C2 alkylene and G1 is —S—S—.In another preferred embodiment, each of Q1 and Q2 is C2 and C5alkylene, respectively, and G1 is —NHC(O)—.

In certain embodiments, f₁, f₂, f₃, f₄, j₁, j₂, j₃ can be 1 and, f₅ andj₄ can be 0, i.e., Q1-G1-Q2-G2-Q3-G3-Q4 are present. In certainembodiments each of Q1, Q2, Q3, and Q4 can be C2 to C8 (e.g. C2, C3, C4,C5, C6, C7, C8) alkylene, G1, G2, and G3 is selected from —SS—,—O—P(O)(OR^(k))—O—, —O—P(S)(OR^(k))—O—, —O—P(S)(SR^(k))—O—,—S—P(O)(OR^(k))—O—, —O—P(O)(OR^(k))—S—, —S—P(O)(OR^(k))—S—,—O—P(S)(OR^(k))—S—, —S—P(S)(OR^(k))-—O—, —O—P(O)(R^(k))—O—,—O—P(S)(R^(k))—O—, —S—P(O)(R^(k))—O—, —S—P(S)(R^(k))—O—,—S—P(O)(R^(k))—S—, —O—P(S)(R^(k))—S—, —C(O)O—, —OC(O)—,—R^(k)C═NNR^(k)—, P1′C(O)NHP2′-, —P1′NHC(O)P2′, NR^(k)C(O)—,—C(O)NR^(k)—, —OC(O)NR^(k)—, —NR^(k)C(O)O—, —O—, —S—, NR^(k)C(O)NR^(k)—,—NR^(k)C(S)NR^(k)—, —NR^(k)C(S)—, —NR^(k)C(S)O—, —C(S)NR^(k)—,—OC(S)NR^(k)—, —NR^(k)C(S)O, —C(O)—, —NR^(k)—. In a preferred embodimenteach of Q1, Q2, Q3, and Q4 is C2 alkylene and G1, G2 and G3 is —S—S— or—NHC(O)NH—.

In certain embodiments, f₁, f₂, f₃, f₄, j₁, j₂, j₃ can be 1 and f₅, andj₄ can be 0, i.e., Q1-G1-Q2-G2-Q3 are present. In certain embodimentseach of Q1, Q2, and Q3, can be C1 to C8 (e.g. C1, C2, C3, C4, C5, C6,C7, C8) alkylene, G1, and G2, can be selected from —SS—,—O—P(O)(OR^(k))—O—, —O—P(S)(OR^(k))—O—, —O—P(S)(SR^(k))—O—,—S—P(O)(OR^(k))—O—, —O—P(O)(OR^(k))—S—, —S—P(O)(OR^(k))—S—,—O—P(S)(OR^(k))—S—, —S—P(S)(OR^(k))—O—, —O—P(O)(R^(k))—O—,—O—P(S)(R^(k))—O—, —S—P(O)(R^(k))—O—, —S—P(S)(R^(k))—O—,—S—P(O)(R^(k))—S—, —O—P(S)(R^(k))—S—, —C(O)O—, —OC(O)—,—R^(k)C═NNR^(k)—, P1′C(O)NHP2′-, —P1′NHC(O)P2′, NR^(k)C(O)—,—C(O)NR^(k)—, —OC(O)NR^(k)—, —NR^(k)C(O)O—, —O—, —S—, NR^(k)C(O)NR^(k)—,—NR^(k)C(S)NR^(k)—, —NR^(k)C(S)—, —NR^(k)C(S)O—, —C(S)NR^(k)—,—OC(S)NR^(k)—, —NR^(k)C(S)O, —C(O)—, —NR^(k)—. In a preferred embodimenteach of Q1, Q2, Q3, and Q4 is C2 or C6 alkylene and G1 or G2 is —S—S— or—NHC(O)—.

In certain embodiments, one of G¹ and G² can be —S—S—, —O—P(O)(OH)—O—,—O—P(S)(OH)—O—, —O—P(S)(SH)—O—, —S—P(O)(OH)—O—, —O—P(O)(OH)—S—,—S—P(O)(OH)—S—, —O—P(S)(OH)—S—, —S—P(S)(OH)—O—, —O—P(O)(H)—O—,—O—P(S)(H)—O—, —S—P(O)(H)—O—, —S—P(S)(H)—O—, —S—P(O)(H)—S—,—O—P(S)(H)—S—, —C═NN—, C(O)O, —OC(O), P1′C(O)NHP2′-, or —P1′NHC(O)P2′and the other —NHC(O)O—, —O—, —C(O)NH—, —S—, —C(S)NH—, carbonyl, —C(O)—,or —NH—.

Exemplary tethers include

E′ Δ E″ —OC(O)NH— —CH₂—CH₂—S—S—CH₂—CH₂— —NHC(O)N— —OC(O)NH——CH₂—CH₂—S—S—CH₂—CH₂—NHC(O)NH—CH₂—CH₂—S—S—CH₂—CH₂— —NHC(O)N— —OC(O)NH——CH₂—CH₂—S—S—CH₂—CH₂—NHC(O)—CH₂—CH₂—CH₂—CH₂— —C(O)N— —C(O)NH——CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—NHC(O)—CH₂—CH₂—S—S—CH₂—CH₂— —C(O)N— —S—S——CH₂—CH₂—NHC(O)—CH₂—CH₂—CH₂—CH₂—CH₂— —C(O)N—

Tethered Ligands

A wide variety of entities, e.g., ligands, can be tethered to an iRNAagent, e.g., to the carrier of a ligand-conjugated monomer subunit.Examples are described below in the context of a ligand-conjugatedmonomer subunit but that is only preferred, entities can be coupled atother points to an iRNA agent.

Preferred moieties are ligands, which are coupled, preferablycovalently, either directly or indirectly via an intervening tether, tothe carrier. In preferred embodiments, the ligand is attached to thecarrier via an intervening tether. As discussed above, the ligand ortethered ligand may be present on the ligand-conjugated monomer\when theligand-conjugated monomer is incorporated into the growing strand. Insome embodiments, the ligand may be incorporated into a “precursor”ligand-conjugated monomer subunit after a “precursor” ligand-conjugatedmonomer subunit has been incorporated into the growing strand. Forexample, a monomer having, e.g., an amino-terminated tether, e.g.,TAP-(CH₂)_(n)NH₂ may be incorporated into a growing sense or antisensestrand. In a subsequent operation, i.e., after incorporation of theprecursor monomer subunit into the strand, a ligand having anelectrophilic group, e.g., a pentafluorophenyl ester or aldehyde group,can subsequently be attached to the precursor ligand-conjugated monomerby coupling the electrophilic group of the ligand with the terminalnucleophilic group of the precursor ligand-conjugated monomer subunittether.

In preferred embodiments, a ligand alters the distribution, targeting orlifetime of an iRNA agent into which it is incorporated. In preferredembodiments a ligand provides an enhanced affinity for a selectedtarget, e.g., molecule, cell or cell type, compartment, e.g., a cellularor organ compartment, tissue, organ or region of the body, as, e.g.,compared to a species absent such a ligand.

Preferred ligands can improve transport, hybridization, and specificityproperties and may also improve nuclease resistance of the resultantnatural or modified oligoribonucleotide, or a polymeric moleculecomprising any combination of monomers described herein and/or naturalor modified ribonucleotides.

Ligands in general can include therapeutic modifiers, e.g., forenhancing uptake; diagnostic compounds or reporter groups e.g., formonitoring distribution; cross-linking agents; nuclease-resistanceconferring moieties; and natural or unusual nucleobases. Generalexamples include lipophiles, lipids, steroids (e.g., uvaol, hecigenin,diosgenin), terpenes (e.g., triterpenes, e.g., sarsasapogenin,Friedelin, epifriedelanol derivatized lithocholic acid), vitamins (e.g.,folic acid, vitamin A, biotin, pyridoxal), carbohydrates, proteins,protein binding agents, integrin targeting molecules, polycationics,peptides, polyamines, and peptide mimics.

Ligands can include a naturally occurring substance, (e.g., human serumalbumin (HSA), low-density lipoprotein (LDL), or globulin); carbohydrate(e.g., a dextran, pullulan, chitin, chitosan, inulin, cyclodextrin orhyaluronic acid); amino acid, or a lipid. The ligand may also be arecombinant or synthetic molecule, such as a synthetic polymer, e.g., asynthetic polyamino acid. Examples of polyamino acids include polyaminoacid is a polylysine (PLL), poly L-aspartic acid, poly L-glutamic acid,styrene-maleic acid anhydride copolymer, poly(L-lactide-co-glycolied)copolymer, divinyl ether-maleic anhydride copolymer,N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol(PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacryllicacid), N-isopropylacrylamide polymers, or polyphosphazine. Example ofpolyamines include: polyethylenimine, polylysine (PLL), spermine,spermidine, polyamine, pseudopeptide-polyamine, peptidomimeticpolyamine, dendrimer polyamine, arginine, amidine, protamine, cationicmoieties, e.g., cationic lipid, cationic porphyrin, quaternary salt of apolyamine, or an alpha helical peptide.

Ligands can also include targeting groups, e.g., a cell or tissuetargeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g.,an antibody, that binds to a specified cell type such as a kidney cell.A targeting group can be a thyrotropin, melanotropin, lectin,glycoprotein, surfactant protein A, Mucin carbohydrate, multivalentlactose, multivalent galactose, N-acetyl-galactosamine,N-acetyl-gulucosamine multivalent mannose, multivalent fucose,glycosylated polyaminoacids, multivalent galactose, transferrin,bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, asteroid, bile acid, folate, vitamin B12, biotin, or an RGD peptide orRGD peptide mimetic.

Other examples of ligands include dyes, intercalating agents (e.g.acridines), cross-linkers (e.g. psoralene, mitomycin C), porphyrins(TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g.,phenazine, dihydrophenazine), artificial endonucleases (e.g. EDTA),lipophilic molecules, e.g., cholesterol, cholic acid, adamantane aceticacid, 1-pyrene butyric acid, dihydrotestosterone, glycerol (e.g., estersand ethers thereof, e.g., C₁₀, C₁₁, C₁₂, C₁₃, C₁₄, C₁₅, C₁₆, C₁₇, C₁₈,C₁₉, or C₂₀ alkyl; e.g., 1,3-bis-O(hexadecyl)glycerol,1,3-bis-O(octaadecyl)glycerol), geranyloxyhexyl group,hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group,palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid,O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine) and peptideconjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents,phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]₂,polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes,haptens (e.g. biotin), transport/absorption facilitators (e.g., aspirin,vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole,bisimidazole, histamine, imidazole clusters, acridine-imidazoleconjugates, Eu3+ complexes of tetraazamacrocycles), dinitrophenyl, HRP,or AP.

Ligands can be proteins, e.g., glycoproteins, or peptides, e.g.,molecules having a specific affinity for a co-ligand, or antibodiese.g., an antibody, that binds to a specified cell type such as a cancercell, endothelial cell, or bone cell. Ligands may also include hormonesand hormone receptors. They can also include non-peptidic species, suchas lipids, lectins, carbohydrates, vitamins, cofactors, multivalentlactose, multivalent galactose, N-acetyl-galactosamine,N-acetyl-gulucosamine multivalent mannose, or multivalent fucose. Theligand can be, for example, a lipopolysaccharide, an activator of p38MAP kinase, or an activator of NF-κB.

The ligand can be a substance, e.g., a drug, which can increase theuptake of the iRNA agent into the cell, for example, by disrupting thecell's cytoskeleton, e.g., by disrupting the cell's microtubules,microfilaments, and/or intermediate filaments. The drug can be, forexample, taxon, vincristine, vinblastine, cytochalasin, nocodazole,japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, ormyoservin.

The ligand can increase the uptake of the iRNA agent into the cell byactivating an inflammatory response, for example. Exemplary ligands thatwould have such an effect include tumor necrosis factor alpha(TNFalpha), interleukin-1 beta, or gamma interferon.

In one aspect, the ligand is a lipid or lipid-based molecule. Such alipid or lipid-based molecule preferably binds a serum protein, e.g.,human serum albumin (HSA). An HSA binding ligand allows for distributionof the conjugate to a target tissue, e.g., a non-kidney target tissue ofthe body. For example, the target tissue can be the liver, includingparenchymal cells of the liver. Other molecules that can bind HSA canalso be used as ligands. For example, neproxin or aspirin can be used. Alipid or lipid-based ligand can (a) increase resistance to degradationof the conjugate, (b) increase targeting or transport into a target cellor cell membrane, and/or (c) can be used to adjust binding to a serumprotein, e.g., HSA.

A lipid based ligand can be used to modulate, e.g., control the bindingof the conjugate to a target tissue. For example, a lipid or lipid-basedligand that binds to HSA more strongly will be less likely to betargeted to the kidney and therefore less likely to be cleared from thebody. A lipid or lipid-based ligand that binds to HSA less strongly canbe used to target the conjugate to the kidney.

In a preferred embodiment, the lipid based ligand binds HSA. Preferably,it binds HSA with a sufficient affinity such that the conjugate will bepreferably distributed to a non-kidney tissue. However, it is preferredthat the affinity not be so strong that the HSA-ligand binding cannot bereversed.

In another preferred embodiment, the lipid based ligand binds HSA weaklyor not at all, such that the conjugate will be preferably distributed tothe kidney. Other moieties that target to kidney cells can also be usedin place of or in addition to the lipid based ligand.

In another aspect, the ligand is a moiety, e.g., a vitamin, which istaken up by a target cell, e.g., a proliferating cell. These areparticularly useful for treating disorders characterized by unwantedcell proliferation, e.g., of the malignant or non-malignant type, e.g.,cancer cells. Exemplary vitamins include vitamin A, E, and K. Otherexemplary vitamins include are B vitamin, e.g., folic acid, B12,riboflavin, biotin, pyridoxal or other vitamins or nutrients taken up bycancer cells. Also included are HSA and low density lipoprotein (LDL).

In another aspect, the ligand is a cell-permeation agent, preferably ahelical cell-permeation agent. Preferably, the agent is amphipathic. Anexemplary agent is a peptide such as tat or antennopedia. If the agentis a peptide, it can be modified, including a peptidylmimetic,invertomers, non-peptide or pseudo-peptide linkages, and use of D-aminoacids. The helical agent is preferably an alpha-helical agent, whichpreferably has a lipophilic and a lipophobic phase.

Peptides that target markers enriched in proliferating cells can beused. E.g., RGD containing peptides and petomimetics can target cancercells, in particular cells that exhibit an α_(v)β₃ integrin. Thus, onecould use RGD peptides, cyclic peptides containing RGD, RGD peptidesthat include D-amino acids, as well as synthetic RGD mimics. In additionto RGD, one can use other moieties that target the α_(v)-β₃ integrinligand. Generally, such ligands can be used to control proliferatingcells and angiogeneis. Preferred conjugates of this type include an iRNAagent that targets PECAM-1, VEGF, or other cancer gene, e.g., a cancergene described herein.

The iRNA agents of the invention are particularly useful when targetedto the liver. An iRNA agent can be targeted to the liver byincorporation of a monomore derivitzed with a ligand which targets tothe liver. For example, a liver-targeting agent can be a lipophilicmoiety. Preferred lipophilic moieties include lipid, cholesterols,oleyl, retinyl, or cholesteryl residues. Other lipophilic moieties thatcan function as liver-targeting agents include cholic acid, adamantaneacetic acid, 1-pyrene butyric acid, dihydrotestosterone,1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol,borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid,myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid,dimethoxytrityl, or phenoxazine.

An iRNA agent can also be targeted to the liver by association with alow-density lipoprotein (LDL), such as lactosylated LDL. Polymericcarriers complexed with sugar residues can also function to target iRNAagents to the liver.

A targeting agent that incorporates a sugar, e.g., galactose and/oranalogues thereof, is particularly useful. These agents target, inparticular, the parenchymal cells of the liver. For example, a targetingmoiety can include more than one or preferably two or three galactosemoieties, spaced about 15 angstroms from each other. The targetingmoiety can alternatively be lactose (e.g., three lactose moieties),which is glucose coupled to a galactose. The targeting moiety can alsobe N-Acetyl-Galactosamine, N-Ac-Glucosamine. A mannose ormannose-6-phosphate targeting moiety can be used for macrophagetargeting.

The ligand can be a peptide or peptidomimetic. A peptidomimetic (alsoreferred to herein as an oligopeptidomimetic) is a molecule capable offolding into a defined three-dimensional structure similar to a naturalpeptide. The attachment of peptide and peptidomimetics to iRNA agentscan affect pharmacokinetic distribution of the iRNA, such as byenhancing cellular recognition and absorption. The peptide orpeptidomimetic moiety can be about 5-50 amino acids long, e.g., about 5,10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long (see Table 1, forexample).

TABLE 1 Exemplary Cell Permeation Peptides. Cell Permeation PeptideAmino acid Sequence Reference Penetratin RQIKIWFQNRRMKWKK (SEQ IDDerossi et al., NO: 1) J. Biol. Chem. 269: 10444, 1994 TatGRKKRRQRRRPPQC (SEQ ID NO: 2) Vives et al., J. fragment (48-60) Biol.Chem., 272: 16010, 1997 Signal GALFLGWLGAAGSTMGAWSQPKKKRKV Chaloin etal., Sequence- (SEQ ID NO: 3) Biochem. Biophys. based peptide Res.Commun., 243: 601, 1998 PVEC LLIILRRRIRKQAHAHSK (SEQ ID Elmquist et NO:4) al., Exp. Cell Res., 269: 237, 2001 TransportanGWTLNSAGYLLKINLKALAALAKKIL Pooga et al., (SEQ ID NO: 5) FASEB J., 12:67, 1998 Amphiphilic KLALKLALKALKAALKLA (SEQ ID Oehlke et al., model NO:6) Mol. Ther., 2: 339, peptide 2000 Arg₉ RRRRRRRRR (SEQ ID NO: 7)Mitchell et al., J. Pept. Res., 56: 318, 2000 Bacterial KFFKFFKFFK (SEQID NO: 8) cell wall permeating LL-37LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES (SEQ ID NO: 9) CecropinSWLSKTAKKLENSAKKRISEGIAIAIQGGPR P1 (SEQ ID NO: 10) α-ACYCRIPACIAGERRYGTCIYQGRLWAFCC defensin (SEQ ID NO: 11) b-DHYNCVSSGGQCLYSACPIFTKIQGTCYRGKAKCCK defensin (SEQ ID NO: 12) BactenecinRKCRIVVIRVCR (SEQ ID NO: 13) PR-39RRRPRPPYLPRPRPPPFFPPRLPPRIPPGFPPRFPPRFPGKR-NH2 (SEQ ID NO: 14)Indolicidin ILPWKWPWWPWRR-NH2 (SEQ ID NO: 15)

A peptide or peptidomimetic can be, for example, a cell permeationpeptide, cationic peptide, amphipathic peptide, or hydrophobic peptide(e.g., consisting primarily of Tyr, Trp or Phe). The peptide moiety canbe a dendrimer peptide, constrained peptide or crosslinked peptide. Inanother alternative, the peptide moiety can include a hydrophobicmembrane translocation sequence (MTS). An exemplary hydrophobicMTS-containing peptide is RFGF having the amino acid sequenceAAVALLPAVLLALLAP (SEQ ID NO:16). An RFGF analogue (e.g., amino acidsequence AALLPVLLAAP (SEQ ID NO:17)) containing a hydrophobic MTS canalso be a targeting moiety. The peptide moiety can be a “delivery”peptide, which can carry large polar molecules including peptides,oligonucleotides, and protein across cell membranes. For example,sequences from the HIV Tat protein (GRKKRRQRRRPPQ (SEQ ID NO:18)) andthe Drosophila Antennapedia protein (RQIKIWFQNRRMKWKK (SEQ ID NO:19))have been found to be capable of functioning as delivery peptides. Apeptide or peptidomimetic can be encoded by a random sequence of DNA,such as a peptide identified from a phage-display library, orone-bead-one-compound (OBOC) combinatorial library (Lam et al., Nature,354:82-84, 1991). Preferably the peptide or peptidomimetic tethered toan iRNA agent via an incorporated monomer unit is a cell targetingpeptide such as an arginine-glycine-aspartic acid (RGD)-peptide, or RGDmimic. A peptide moiety can range in length from about 5 amino acids toabout 40 amino acids. The peptide moieties can have a structuralmodification, such as to increase stability or direct conformationalproperties. Any of the structural modifications described below can beutilized.

An RGD peptide moiety can be used to target a tumor cell, such as anendothelial tumor cell or a breast cancer tumor cell (Zitzmann et al.,Cancer Res., 62:5139-43, 2002). An RGD peptide can facilitate targetingof an iRNA agent to tumors of a variety of other tissues, including thelung, kidney, spleen, or liver (Aoki et al., Cancer Gene Therapy8:783-787, 2001). Preferably, the RGD peptide will facilitate targetingof an iRNA agent to the kidney. The RGD peptide can be linear or cyclic,and can be modified, e.g., glycosylated or methylated to facilitatetargeting to specific tissues. For example, a glycosylated RGD peptidecan deliver an iRNA agent to a tumor cell expressing α_(v)β₃ (Haubner etal., Jour. Nucl. Med., 42:326-336, 2001).

Peptides that target markers enriched in proliferating cells can beused. E.g., RGD containing peptides and peptidomimetics can targetcancer cells, in particular cells that exhibit an α_(v)β₃ integrin.Thus, one could use RGD peptides, cyclic peptides containing RGD, RGDpeptides that include D-amino acids, as well as synthetic RGD mimics. Inaddition to RGD, one can use other moieties that target the α_(v)-β₃integrin ligand. Generally, such ligands can be used to controlproliferating cells and angiogeneis. Preferred conjugates of this typeinclude an iRNA agent that targets PECAM-1, VEGF, or other cancer gene,e.g., a cancer gene described herein.

A “cell permeation peptide” is capable of permeating a cell, e.g., amicrobial cell, such as a bacterial or fungal cell, or a mammalian cell,such as a human cell. A microbial cell-permeating peptide can be, forexample, an α-helical linear peptide (e.g., LL-37 or Ceropin P1), adisulfide bond-containing peptide (e.g., α-defensin, β-defensin orbactenecin), or a peptide containing only one or two dominating aminoacids (e.g., PR-39 or indolicidin). A cell permeation peptide can alsoinclude a nuclear localization signal (NLS). For example, a cellpermeation peptide can be a bipartite amphipathic peptide, such as MPG,which is derived from the fusion peptide domain of HIV-1 gp41 and theNLS of SV40 large T antigen (Simeoni et al., Nucl. Acids Res.31:2717-2724, 2003).

In one embodiment, a targeting peptide tethered to an ligand-conjugatedmonomer can be an amphipathic α-helical peptide. Exemplary amphipathicα-helical peptides include, but are not limited to, cecropins,lycotoxins, paradaxins, buforin, CPF, bombinin-like peptide (BLP),cathelicidins, ceratotoxins, S. clava peptides, hagfish intestinalantimicrobial peptides (HFIAPs), magainines, brevinins-2, dermaseptins,melittins, pleurocidin, H₂A peptides, Xenopus peptides, esculentinis-1,and caerins. A number of factors will preferably be considered tomaintain the integrity of helix stability. For example, a maximum numberof helix stabilization residues will be utilized (e.g., leu, ala, orlys), and a minimum number helix destabilization residues will beutilized (e.g., proline, or cyclic monomeric units. The capping residuewill be considered (for example Gly is an exemplary N-capping residueand/or C-terminal amidation can be used to provide an extra H-bond tostabilize the helix. Formation of salt bridges between residues withopposite charges, separated by i±3, or i±4 positions can providestability. For example, cationic residues such as lysine, arginine,homo-arginine, ornithine or histidine can form salt bridges with theanionic residues glutamate or aspartate.

Peptide and petidomimetic ligands include those having naturallyoccurring or modified peptides, e.g., D or L peptides; α, β, or γpeptides; N-methyl peptides; azapeptides; peptides having one or moreamide, i.e., peptide, linkages replaced with one or more urea, thiourea,carbamate, or sulfonyl urea linkages; or cyclic peptides.

In some embodiments, the ligand can be any of the nucleobases describedherein.

In some embodiments, the ligand can be a substituted amine, e.g.dimethylamino. In certain embodiments the substituted amine can berendered cationic, e.g., by quaternization, e.g., protonation oralkylation. In certain embodiments, the substituted amine can be at theterminal position of a relatively hydrophobic chain, e.g., an alkylenechain.

In some embodiments, the ligand can be one of the following triterpenes:

In some embodiments, the ligand can be substituted or unsubstitutedcholesterol, or a stereoisomer thereof or one of the following steroids:

Methods for Making iRNA Agents

A listing of ribonucleosides containing the unusual bases describedherein are described online in “The RNA Modification Database”maintained by Pamela F. Crain, Jef Rozenski and James A. McCloskey;Departments of Medicinal Chemistry and Biochemistry, University of Utah,Salt Lake City, Utah 84112, USA.

The 5′ silyl protecting group can be used in conjunction with acidlabile orthoesters at the 2′ position of ribonucleosides to synthesizeoligonucleotides via phosphoramidite chemistry. Final deprotectionconditions are known not to significantly degrade RNA products.Functional groups on the unusual and universal bases are blocked duringoligonucleotide synthesis with protecting groups that are compatiblewith the operations being performed that are described herein. Allsyntheses can be can be conducted in any automated or manual synthesizeron large, medium, or small scale. The syntheses may also be carried outin multiple well plates or glass slides.

The 5′-O-silyl group can be removed via exposure to fluoride ions, whichcan include any source of fluoride ion, e.g., those salts containingfluoride ion paired with inorganic counterions e.g., cesium fluoride andpotassium fluoride or those salts containing fluoride ion paired with anorganic counterion, e.g., a tetraalkylammonium fluoride. A crown ethercatalyst can be utilized in combination with the inorganic fluoride inthe deprotection reaction. Preferred fluoride ion source aretetrabutylammonium fluoride or aminehydrofluorides (e.g., combiningaqueous HF with triethylamine in a dipolar aprotic solvent, e.g.,dimethylformamide).

The choice of protecting groups for use on the phosphite triesters andphosphotriesters can alter the stability of the triesters towardsfluoride. Methyl protection of the phosphotriester or phosphitetriestercan stabilize the linkage against fluoride ions and improve processyields.

Since ribonucleosides have a reactive 2′ hydroxyl substituent, it can bedesirable to protect the reactive 2′ position in RNA with a protectinggroup that is compatible with a 5′-O-silyl protecting group, e.g. onestable to fluoride. Orthoesters meet this criterion and can be readilyremoved in a final acid deprotection step that can result in minimal RNAdegradation.

Tetrazole catalysts can be used in the standard phosphoramidite couplingreaction. Preferred catalysts include e.g. tetrazole, S-ethyl-tetrazole,p-nitrophenyltetrazole.

The general process is as follows. Nucleosides are suitably protectedand functionalized for use in solid-phase or solution-phase synthesis ofRNA oligonucleotides. The 2′-hydroxyl group in a ribonucleotide can bemodified using a tris orthoester reagent. The 2′-hydroxyl can bemodified to yield a 2′-O-orthoester nucleoside by reacting theribonucleoside with the tris orthoester reagent in the presence of anacidic catalyst, e.g., pyridinium p-toluene sulfonate. This reaction isknown to those skilled in the art. The product can then be subjected tofurther protecting group reactions (e.g., 5′-O-silylation) andfunctionalizations (e.g., 3′-O-phosphitylation) to produce a desiredreagent (e.g., nucleoside phosphoramidite) for incorporation within anoligonucleotide or polymer by reactions known to those skilled in theart.

Preferred orthoesters include those comprising ethylene glycol ligandswhich are protected with acyl or ester protecting groups. Specifically,the preferred acyl group is acetyl. The nucleoside reagents may then beused by those skilled in the art to synthesize RNA oligonucleotides oncommercially available synthesizer instruments, e.g. Gene Assembler Plus(Pharmacia), 380B (Applied Biosystems). Following synthesis (eithersolution-phase or solid-phase) of an oligonucleotide or polymer, theproduct can be subjected to one or more reactions using non-acidicreagents. One of these reactions may be strong basic conditions, forexample, 40% methylamine in water for 10 minutes at 55.degree. C., whichwill remove the acyl protecting groups from the ethylene glycol ligandsbut leave the orthoester moiety attached. The resultant orthoester maybe left attached when the polymer or oligonucleotide is used insubsequent applications, or it may be removed in a final mildly-acidicreaction, for example, 10 minutes at 55.degree. C. in 50 mM acetic acid,pH 3.0, followed by addition of equal volume of 150 mM TRIS buffer for10 minutes at 55.degree. C.

Universal bases are described in “Survey and Summary: The Applicationsof Universal DNA base analogues” Loakes, D., Nucleic Acid Research 2001,29, 2437, which is incorporated by reference in its entirety. Specificexamples are described in the following: Liu, D.; Moran, S.; Kool, E. T.Chem. Biol., 1997, 4, 919-926; Morales, J. C.; Kool, E. T. Biochemistry,2000, 39, 2626-2632; Matray, T, J.; Kool, E. T. J. Am. Chem. Soc., 1998,120, 6191-6192; Moran, S. Ren, R. X.-F.; Rumney I V, S.; Kool, E. T. J.Am. Chem. Soc., 1997, 119, 2056-2057; Guckian, K. M.; Morales, J. C.;Kool, E. T. J. Org. Chem., 1998, 63, 9652-9656; Berger, M.; Wu. Y.;Ogawa, A. K.; McMinn, D. L.; Schultz, P. G.; Romesberg, F. E. NucleicAcids Res., 2000, 28, 2911-2914; Ogawa, A. K.; Wu, Y.; McMinn, D. L.;Liu, J.; Schultz, P. G.; Romesberg, F. E. J. Am. Chem. Soc., 2000, 122,3274-3287; Ogawa, A. K.; Wu. Y.; Berger, M.; Schultz, P. G.; Romesberg,F. E. J. Am. Chem. Soc., 2000, 122, 8803-8804; Tae, E. L.; Wu, Y.; Xia,G.; Schultz, P. G.; Romesberg, F. E. J. Am. Chem. Soc., 2001, 123,7439-7440; Wu, Y.; Ogawa, A. K.; Berger, M.; McMinn, D. L.; Schultz, P.G.; Romesberg, F. E. J. Am. Chem. Soc., 2000, 122, 7621-7632; McMinn, D.L.; Ogawa. A. K.; Wu, Y.; Liu, J.; Schultz, P. G.; Romesberg, F. E. J.Am. Chem. Soc., 1999, 121, 11585-11586; Brotschi, C.; Haberli, A.;Leumann, C, J. Angew. Chem. Int. Ed., 2001, 40, 3012-3014; Weizman, H.;Tor, Y. J. Am. Chem. Soc., 2001, 123, 3375-3376; Lan, T.; McLaughlin, L.W. J. Am. Chem. Soc., 2000, 122, 6512-13.

As discussed above, the monomers and methods described herein can beused in the preparation of modified RNA molecules, or polymericmolecules comprising any combination of monomer compounds describedherein and/or natural or modified ribonucleotides in which one or moresubunits contain an unusual or universal base. Modified RNA moleculesinclude e.g. those molecules containing a chemically or stereochemicallymodified nucleoside (e.g., having one or more backbone modifications,e.g., phosphorothioate or P-alkyl; having one or more sugarmodifications, e.g., 2′-OCH₃ or 2′-F; and/or having one or more basemodifications, e.g., 5-alkylamino or 5-allylamino) or a nucleosidesurrogate.

Coupling of 5′-hydroxyl groups with phosphoramidites forms phosphiteester intermediates, which in turn are oxidized e.g., with iodine, tothe phosphate diester. Alternatively, the phosphites may be treated withe.g., sulfur, selenium, amino, and boron reagents to form modifiedphosphate backbones. Linkages between the monomers described herein anda nucleoside or oligonucleotide chain can also be treated with iodine,sulfur, selenium, amino, and boron reagents to form unmodified andmodified phosphate backbones respectively. Similarly, the monomersdescribed herein may be coupled with nucleosides or oligonucleotidescontaining any of the modifications or nucleoside surrogates describedherein.

The synthesis and purification of oligonucleotide peptide conjugates canbe performed by established methods. See, for example, Trufert et al.,Tetrahedron, 52:3005, 1996; and Manoharan, “Oligonucleotide Conjugatesin Antisense Technology,” in Antisense Drug Technology, ed. S. T.Crooke, Marcel Dekker, Inc., 2001. Exemplary methods are shown in FIGS.4 and 5.

In one embodiment of the invention, a peptidomimetic can be modified tocreate a constrained peptide that adopts a distinct and specificpreferred conformation, which can increase the potency and selectivityof the peptide. For example, the constrained peptide can be anazapeptide (Gante, Synthesis, 405-413, 1989). An azapeptide issynthesized by replacing the α-carbon of an amino acid with a nitrogenatom without changing the structure of the amino acid side chain. Forexample, the azapeptide can be synthesized by using hydrazine intraditional peptide synthesis coupling methods, such as by reactinghydrazine with a “carbonyl donor,” e.g., phenylchloroformate. A generalazapeptide synthesis is shown in FIG. 6.

In one embodiment of the invention, a peptide or peptidomimetic (e.g., apeptide or peptidomimetic tethered to an ligand-conjugated monomer) canbe an N-methyl peptide. N-methyl peptides are composed of N-methyl aminoacids, which provide an additional methyl group in the peptide backbone,thereby potentially providing additional means of resistance toproteolytic cleavage. N-methyl peptides can by synthesized by methodsknown in the art (see, for example, Lindgren et al., Trends Pharmacol.Sci. 21:99, 2000; Cell Penetrating Peptides: Processes and Applications,Langel, ed., CRC Press, Boca Raton, Fla., 2002; Fische et al.,Bioconjugate. Chem. 12: 825, 2001; Wander et al., J. Am. Chem. Soc.,124:13382, 2002). For example, an Ant or Tat peptide can be an N-methylpeptide. An exemplary synthesis is shown in FIG. 7.

In one embodiment of the invention, a peptide or peptidomimetic (e.g., apeptide or peptidomimetic tethered to a ligand-conjugated monomer) canbe β-peptide. β-peptides form stable secondary structures such ashelices, pleated sheets, turns and hairpins in solutions. Their cyclicderivatives can fold into nanotubes in the solid state. β-peptides areresistant to degradation by proteolytic enzymes. β-peptides can besynthesized by methods known in the art. For example, an Ant or Tatpeptide can be a β-peptide. An exemplary synthesis is shown in FIG. 8.

In one embodiment of the invention, a peptide or peptidomimetic (e.g., apeptide or peptidomimetic tethered to a ligand-conjugated monomer) canbe a oligocarbamate. Oligocarbamate peptides are internalized into acell by a transport pathway facilitated by carbamate transporters. Forexample, an Ant or Tat peptide can be an oligocarbamate. An exemplarysynthesis is shown in FIG. 9.

In one embodiment of the invention, a peptide or peptidomimetic (e.g., apeptide or peptidomimetic tethered to a ligand-conjugated monomer) canbe an oligourea conjugate (or an oligothiourea conjugate), in which theamide bond of a peptidomimetic is replaced with a urea moiety.Replacement of the amide bond provides increased resistance todegradation by proteolytic enzymes, e.g., proteolytic enzymes in thegastrointestinal tract. In one embodiment, an oligourea conjugate istethered to an iRNA agent for use in oral delivery. The backbone in eachrepeating unit of an oligourea peptidomimetic can be extended by onecarbon atom in comparison with the natural amino acid. The single carbonatom extension can increase peptide stability and lipophilicity, forexample. An oligourea peptide can therefore be advantageous when an iRNAagent is directed for passage through a bacterial cell wall, or when aniRNA agent must traverse the blood-brain barrier, such as for thetreatment of a neurological disorder. In one embodiment, a hydrogenbonding unit is conjugated to the oligourea peptide, such as to createan increased affinity with a receptor. For example, an Ant or Tatpeptide can be an oligourea conjugate (or an oligothiourea conjugate).An exemplary synthesis is shown in FIG. 10.

The siRNA peptide conjugates of the invention can be affiliated with,e.g., tethered to, ligand-conjugated monomers occurring at variouspositions on an iRNA agent. For example, a peptide can be terminallyconjugated, on either the sense or the antisense strand, or a peptidecan be bisconjugated (one peptide tethered to each end, one conjugatedto the sense strand, and one conjugated to the antisense strand). Inanother option, the peptide can be internally conjugated, such as in theloop of a short hairpin iRNA agent. In yet another option, the peptidecan be affiliated with a complex, such as a peptide-carrier complex.

A peptide-carrier complex consists of at least a carrier molecule, whichcan encapsulate one or more iRNA agents (such as for delivery to abiological system and/or a cell), and a peptide moiety tethered to theoutside of the carrier molecule, such as for targeting the carriercomplex to a particular tissue or cell type. A carrier complex can carryadditional targeting molecules on the exterior of the complex, orfusogenic agents to aid in cell delivery. The one or more iRNA agentsencapsulated within the carrier can be conjugated to lipophilicmolecules, which can aid in the delivery of the agents to the interiorof the carrier.

A carrier molecule or structure can be, for example, a micelle, aliposome (e.g., a cationic liposome), a nanoparticle, a microsphere, ora biodegradable polymer. A peptide moiety can be tethered to the carriermolecule by a variety of linkages, such as a disulfide linkage, an acidlabile linkage, a peptide-based linkage, an oxyamino linkage or ahydrazine linkage. For example, a peptide-based linkage can be a GFLGpeptide. Certain linkages will have particular advantages, and theadvantages (or disadvantages) can be considered depending on the tissuetarget or intended use. For example, peptide based linkages are stablein the blood stream but are susceptible to enzymatic cleavage in thelysosomes. A schematic of preferred carriers is shown in FIG. 11.

The protected monomer compounds can be separated from a reaction mixtureand further purified by a method such as column chromatography, highpressure liquid chromatography, or recrystallization. As can beappreciated by the skilled artisan, further methods of synthesizing thecompounds of the formulae herein will be evident to those of ordinaryskill in the art. Additionally, the various synthetic steps may beperformed in an alternate sequence or order to give the desiredcompounds. Other synthetic chemistry transformations, protecting groups(e.g., for hydroxyl, amino, etc. present on the bases) and protectinggroup methodologies (protection and deprotection) useful in synthesizingthe compounds described herein are known in the art and include, forexample, those such as described in R. Larock, Comprehensive OrganicTransformations, VCH Publishers (1989); T. W. Greene and P. G. M. Wuts,Protective Groups in Organic Synthesis, 2d. Ed., John Wiley and Sons(1991); L. Fieser and M. Fieser, Fieser and Fieser's Reagents forOrganic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed.,Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons(1995), and subsequent editions thereof.

The protected monomer compounds of this invention may contain one ormore asymmetric centers and thus occur as racemates and racemicmixtures, single enantiomers, individual diastereomers anddiastereomeric mixtures. All such isomeric forms of these compounds areexpressly included in the present invention. The compounds describedherein can also contain linkages (e.g., carbon-carbon bonds,carbon-nitrogen bonds, e.g., amides) or substituents that can restrictbond rotation, e.g. restriction resulting from the presence of a ring ordouble bond. Accordingly, all cis/trans, E/Z isomers, and rotationalisomers (rotamers) are expressly included herein. The compounds of thisinvention may also be represented in multiple tautomeric forms, in suchinstances, the invention expressly includes all tautomeric forms of thecompounds described herein (e.g., alkylation of a ring system may resultin alkylation at multiple sites, the invention expressly includes allsuch reaction products). All such isomeric forms of such compounds areexpressly included in the present invention. All crystal forms of thecompounds described herein are expressly included in the presentinvention.

Representative ligand-conjugated monomers and typical syntheses forpreparing ligand-conjugated monomers and related compounds are describedin U.S. Ser. No. 10/916,185, filed Aug. 10, 2004, which is herebyincorporated by reference. As discussed elsewhere, protecting groups forligand-conjugated monomer hydroxyl groups, e.g., OFG¹, include but arenot limited to the dimethoxytrityl group (DMT). For example, it can bedesirable in some embodiments to use silicon-based protecting groups asa protecting group for OFG¹. Silicon-based protecting groups cantherefore be used in conjunction with or in place of the DMT group asnecessary or desired. Thus, the ligand-conjugated monomers and synthesesdelineated below, which feature the DMT protecting group as a protectinggroup for OFG¹, is not to be construed as limiting in any way to theinvention.

Targeting

The iRNA agents of the invention are particularly useful when targetedto the liver. The chemical modifications described herein can becombined with the compounds and methods described in U.S. ProvisionalApplication 60/462,097, filed on Apr. 9, 2003, which is herebyincorporated by reference; and U.S. Provisional Application 60/461,915,filed on Apr. 10, 2003, which is hereby incorporated by reference. Forexample, an iRNA agent can be targeted to the liver by incorporation ofan RRMS containing a ligand that targets the liver, e.g., a lipophilicmoiety. Preferred lipophilic moieties include lipid, cholesterols,oleyl, retinyl, or cholesteryl residues. Other lipophilic moieties thatcan function as liver-targeting agents include cholic acid, adamantaneacetic acid, 1-pyrene butyric acid, dihydrotestosterone,1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol,borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid,myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid,dimethoxytrityl, or phenoxazine.

An iRNA agent can also be targeted to the liver by association with alow-density lipoprotein (LDL), such as lactosylated LDL. Polymericcarriers complexed with sugar residues can also function to target iRNAagents to the liver.

Conjugation of an iRNA agent with a serum albumin (SA), such as humanserum albumin, can also be used to target the iRNA agent to a non-kidneytissue, such as the liver.

An iRNA agent targeted to the liver by an RRMS targeting moietydescribed herein can target a gene expressed in the liver. For example,the iRNA agent can target p21(WAF1/DIP1), P27(KIP1), the α-fetoproteingene, beta-catenin, or c-MET, such as for treating a cancer of theliver. In another embodiment, the iRNA agent can target apoB-100, suchas for the treatment of an HDL/LDL cholesterol imbalance; dyslipidemias,e.g., familial combined hyperlipidemia (FCHL), or acquiredhyperlipidemia; hypercholesterolemia; statin-resistanthypercholesterolemia; coronary artery disease (CAD); coronary heartdisease (CHD); or atherosclerosis. In another embodiment, the iRNA agentcan target forkhead homologue in rhabdomyosarcoma (FKHR); glucagon;glucagon receptor; glycogen phosphorylase; PPAR-Gamma Coactivator(PGC-1); Fructose-1,6-bisphosphatase; glucose-6-phosphatase;glucose-6-phosphate translocator; glucokinase inhibitory regulatoryprotein; or phosphoenolpyruvate carboxykinase (PEPCK), such as toinhibit hepatic glucose production in a mammal, such as a human, such asfor the treatment of diabetes. In another embodiment, an iRNA agenttargeted to the liver can target Factor V, e.g., the Leiden Factor Vallele, such as to reduce the tendency to form a blood clot. An iRNAagent targeted to the liver can include a sequence which targetshepatitis virus (e.g., Hepatitis A, B, C, D, E, F, G, or H). Forexample, an iRNA agent of the invention can target any one of thenonstructural proteins of HCV: NS3, 4A, 4B, 5A, or 5B. For the treatmentof hepatitis B, an iRNA agent can target the protein X (HBx) gene, forexample.

A targeting agent that incorporates a sugar, e.g., galactose and/oranalogues thereof, can be useful. These agents target, for example, theparenchymal cells of the liver. For example, a targeting moiety caninclude more than one or preferably two or three galactose moieties,spaced about 15 angstroms from each other. The targeting moiety canalternatively be lactose (e.g., three lactose moieties), which isglucose coupled to a galactose. The targeting moiety can also beN-Acetyl-Galactosamine, N-Ac-Glucosamine. A mannose ormannose-6-phosphate targeting moiety can be used for macrophagetargeting.

The iRNA agents of the invention are particularly useful when targetedto the kidney. The chemical modifications described herein can becombined with the compounds and methods described in U.S. ProvisionalApplication 60/460,783, filed on Apr. 3, 2003, which is herebyincorporated by reference; and 60/503,414, filed on Sep. 15, 2003, whichis hereby incorporated by reference. An iRNA agent can be targeted tothe kidney by incorporation of an RRMS containing a ligand that targetsthe kidney.

An iRNA agent targeted to the kidney by an RRMS targeting moietydescribed herein can target a gene expressed in the kidney.

Ligands on RRMSs can include folic acid, glucose, cholesterol, cholicacid, Vitamin E, Vitamin K, or Vitamin A.

Conjugation with a Lipophilic Moiety which Promotes Entry into Cells

RNAi agents can be modified so as to enhance entry into cells, e.g., byconjugation with a lipophilic moiety. A lipophilic moiety can beattached to an RNAi agent in a number of ways but a preferred mode ofattachment is by attachment to an RRMS, e.g., pyrroline-based RRMS. Thelipohilic moiety can be attached at the N atom of a pyrroline-basedRRMS. Examples of lipophilic moieties include cholesterols, lipid,oleyl, retinyl, or cholesteryl residues. Other lipophilic moietiesinclude cholic acid, adamantane acetic acid, 1-pyrene butyric acid,dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexylgroup, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecylgroup, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid,O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine. Cholesterolis a particularly preferred example.

The lipohilic moiety can be attached at the 3′ terminus, the 5′terminus,or internally, preferably on the sense strand. The lipohilic moiety canbe attached to an RRMS, e.g., a pyrroline-based RRMS which is at the 3′terminus, the 5′terminus, or internal, in the sense strand. Theattachment can be direct or through a tethering molecule. Tethers,spacers or linkers discussed herein can be used to attach the moiety tothe RRMS.

An iRNA agent to which one or more lipophilic (e.g., cholesterol)molecules is conjugated (referred to herein as an “iRNA-lipophilicconjugate”) can be delivered in vivo, e.g., to a cell, such as a cell ofa tissue in a subject, such as a mammalian subject (e.g., a human ormouse). Alternatively, or in addition, the iRNA agent can be deliveredin vitro, e.g., to a cell in a cell line. Cell lines can be, forexample, from a vertebrate organism, such as a mammal (e.g., a human ora mouse). Delivery of an iRNA-cholesterol conjugate to a cell line canbe in the absence of other transfection reagents. For example, deliveryof an iRNA-lipophilic conjugate to a cell can be in the absence of, oroptionally, in the presence of, Lipofectamine™ (Invitrogen, Carlsbad,Calif.), Lipofectamine 2000™, TransIT-TKO™ (Mirus, Madison, Wis.),FuGENE 6 (Roche, Indianapolis, Ind.), polyethylenimine, X-tremeGENE Q2(Roche, Indianapolis, Ind.), DOTAP, DOSPER, or Metafectene™ (Biontex,Munich, Germany), or another transfection reagent. Exemplary cell linescan be provided by the American Type Culture Collection (ATCC)(Manassus, Va.). An iRNA-lipophilic conjugate can be delivered to a cellline, such as any cell line described herein, to target a specific genefor downregulation.

In one example, an iRNA-lipophilic conjugate can be delivered to aprimary cell line, e.g., a synoviocyte (such as type B), cardiacmyocyte, keratinocyte, hepatocyte, smooth muscle cell, endothelial cell,or dermal fibroblast cell line.

In another example, an iRNA-lipophilic conjugate can be delivered tomonocyte, or myeloid cell line, e.g., a THP1, Raw264.7, IC21, P388D1,U937, or HL60 cell line.

In another example, an iRNA-lipophilic conjugate can be delivered tolymphoma, or leukemia cell line, e.g., an SEM-K2, WEHI-231, HB56, TIB55,Jurkat, K562, EL4, LRMB, Bcl-1, or TF1 cell line. For example, aniRNA-lipophilic conjugate can be delivered to a lymphoma cell line totarget a specific gene for down regulation. An iRNA-lipophilic agent cantarget (down-regulate) a gene in a Jurkat cell line, for example, thatencodes an immune factor, such as an interleukin gene, e.g., IL-1, IL-2,IL-5, IL-6, IL-8, IL-10, IL-15, IL-16, IL-17, or IL-18. In anotheraspect, an iRNA-lipophilic conjugate can target a gene that encodes areceptor of an interleukin.

An iRNA-lipophilic conjugate can target a gene resulting from achromosomal translocation, such as BCR-ABL, TEL-AML-1, EWS-FLI1,EWS-ERG, TLS-FUS, PAX3-FKHR, or AML1-ETO. For example, aniRNA-lipophilic conjugate that targets a gene resulting from achromosomal translocation can be delivered to a leukemia cell line,e.g., any of the leukemia cell lines discussed above.

An iRNA-lipophilic conjugate can be delivered to an immortalized cellline, including immortalized cell lines from a variety of differenttissue types, including but not limited to T-cells, fibroblast cells,epithelial cells (e.g., kidney epithelial cells) and muscle cells (e.g.,smooth muscle cells). Exemplary immortalized cell lines are CTLL-2(T-cell), Rat 6 (fibroblast), VERO (fibroblast), MRC5 (fibroblast), CV1(fibroblast), Cos7 (fibroblast), RPTE (kidney epithelial), and A10(smooth muscle) cell lines.

An iRNA-lipophilic conjugate can be delivered to a mast cell line, forexample. An iRNA-lipophilic conjugate delivered to a mast cell line cantarget, for example, a gene encoding a GRB2 associated binding protein(e.g., GAB2).

An iRNA-lipophilic conjugate can be delivered to an adherent tumor cellline, including tumor cell lines from a variety of different tissuetypes including but not limited to cancers of the bladder, lung, breast,cervix, colon, pancreas, prostate, and liver, melanomas, andglioblastomas. Exemplary tumor cell lines include the T24 (bladder), J82(bladder), A549 (lung), Calu1 (lung), SW480 (colon), SW620 (colon),CaCo2 (colon), A375 (melanoma), C8161 (melanoma), MCF-7 (breast),MDA-MB-231 (breast), HeLa (cervical), HeLa S3 (cervical), MiaPaCall(pancreas), Panc1 (pancreas), PC-3 (prostate), LNCaP (prostate), HepG2(hepatocellular), and U87 (glioblastoma) cell lines. An iRNA-lipophilicconjugate that targets a specific gene can be delivered to an adherenttumor cell line. For example, an iRNA-lipophilic conjugate that targetsa growth factor or growth factor receptor, such as a TGF-beta (e.g.,TGF-beta 1) or TGF-beta receptor gene, can be delivered to an A549 orHepG2 cell line, a DLD2 colon carcinoma line, or a SKOV3 adenocarcinomacell line. Other exemplary target growth factor genes include plateletderived growth factor (PDGF) and PDGF-Receptor (PDGFR), vascularendothelial growth factor (VEGF) and VEGF receptor genes (e.g., VEGFr1,VEGFr2, or VEGFr3), and insulin-growth factor receptors, such as type Iinsulin-growth factor (IGF) receptors, including IGF-1R, DAF-2 and InR.

In another example, an iRNA-lipophilic conjugate that targets one ormore genes in a protein tyrosine phosphatase type IVA (PRL3, also calledPTP4A3) gene family (e.g., PRL1, PRL2, or PRL3), or a gene in a PRL3pathway, can be delivered to an A549 cell line, or to a culturedcolorectal epithelial cell line.

In another example, an iRNA-lipophilic conjugate can target one or moreprotein kinase C genes in an adherent tumor cell line, such as in amouse Lewis lung carcinoma, B16 melanoma, mouse mammary adenocarcinomaor fibrosarcoma; or a human lung carcinoma, bladder carcinoma,pancreatic cancer, gastric cancer, breast cancer, thyroid carcinoma, ormelanoma. An iRNA-lipophilic conjugate can target a gene encoding a PKCisoforms, such as PKC-alpha, PKC beta I, PKC beta II, PKC gamma, PKCdelta, PKC epsilon, and/or PKC zeta, or a gene encoding one or morereceptors of a protein kinase C polypeptide.

In another example, an iRNA-lipophilic conjugate can target a geneencoding a P-glycoprotein, such as a gene in the multidrug resistance(MDR) gene family, e.g., MDR1. An iRNA-lipophilic conjugate that targetsan MDR gene can be delivered, for example, to a human KB carcinoma cellline, a human leukemia or ovarian carcinoma cell line, or a lungcarcinoma cell line such as A549.

In another example, an iRNA-lipophilic conjugate can target a geneencoding a gene in the telomerase pathway, such as TERT or thetelomerase template RNA (TR/TERC). An iRNA-lipophilic conjugate thattargets a gene in the telomerase pathway can be delivered, for example,to a human cancer cell line, e.g., a breast, cervical, endometrial,meningeal, lung, testicular, or ovarian cancer cell line.

In another example, an iRNA-lipophilic conjugate delivered to anadherent cell line (e.g., a HeLa, parathyroid adenoma, or A549 cellline) can target a cyclin gene, such as cyclin D1.

In another example, an iRNA-lipophilic conjugate delivered to anadherent cell line (e.g., a HeLa cell line) can target an NF-kappaB orREL-A gene, or a gene encoding a ligand or receptor of an NF-kappaB orREL-A polypeptide, or a gene encoding a subunit of NF-kappaB, such asREL-B, REL, NF-kappaB1 or NF-kappaB2.

In another example, an iRNA-lipophilic conjugate delivered to anadherent cell line (e.g., a HeLa or A549 cell line) can target a geneencoding proliferating cell nuclear antigen (PCNA), a checkpoint kinasegene (CHK-1), or a c-fos gene. Further, an iRNA-lipophilic conjugate cantarget any gene in a PCNA, CHK-1, or c-fos pathway. For example aniRNA-lipophilic conjugate can down-regulate a gene encoding jun, whichis in the c-fos pathway.

In another example, an iRNA-lipophilic conjugate delivered to anadherent cell line (e.g., an A549, T24, or A375 cell line) can target agene encoding BCL2.

The cell lines described herein can be used to test iRNA-lipophilicconjugates that target exogenous, such as pathogenic or viral, nucleicacids. For example, an iRNA-lipophilic conjugate that targets ahepatitis viral gene can be delivered to a human hepatoma cell line,such as a HepG2 or Huh cell line, e.g., Huh1, Huh4, Huh7, and the like,that has been infected with the virus (e.g., an HAV, HBV, or HCV). Forexample, an iRNA-lipophilic conjugate that targets an HCV gene, such asin an infected Huh cell line, can target a conserved region of the HCVgenome, such as the 5′-non-coding region (NCR), the 5′ end of the coreprotein coding region, or the 3′-NCR.

The cell lines described herein can be also be used to testiRNA-lipophilic conjugates that target exogenous recombinant nucleicacids, such as reporter genes (e.g., GFP, lacZ, beta-galactosidase, andthe like), that are transfected (transiently or stably) into the celllines.

In one aspect, an iRNA-lipophilic conjugate can be delivered to a B-cellline, e.g., BC-3, C1R, or ARH-77 cells. In another aspect, aniRNA-lipophilic conjugate can be delivered to T-cells, e.g., J45.01,MOLT, and CCRF-CEM cells. An iRNA-lipophilic conjugate can target anendogenous or exogenous nucleic acid. For example, development of aniRNA-lipophilic conjugate that targets an HIV gene can be tested againstan exogenous HIV nucleic acid in a B cell or T cell line, or in amacrophage or endothelial cell culture system.

An iRNA-lipophilic conjugate can be delivered to cells derived fromendoderm, epithelium, or mesoderm. For example, an iRNA-lipophilicconjugate can be delivered to cells of the HeLa or MCF7 epithelial celllines, to cells of the HUVEC endothelial cell line, or to cells of anSK-UT or HASMC mesodermal cell line. In one example, an iRNA-lipophilicagent that targets a TGF-beta nucleic acid or TGF-beta receptor nucleicacid can be delivered to a vascular smooth muscle cell line, e.g., thekidney fibroblast 293 cell line. Other exemplary targets ofiRNA-lipophilic conjugates delivered to fibroblast cells, such as 293cells, included a protein tyrosine phosphatase-1B (PTP-1B) gene or MAPkinase gene (e.g., ERK1, ERK2, JNK1, JNK2, and p38). In another example,an iRNA-lipophilic conjugate that targets an MDR gene fordown-regulation can be delivered to the human intestinal epithelial cellline, Caco-2.

In one example, an iRNA-lipophilic conjugate delivered to a cell line,such as an epithelial or mesodermal cell line (e.g., a HeLa or HASMCcell line, respectively), can target a gene encoding a Myc or Mybpolypeptide, e.g., c-Myc, N-Myc, L-Myc, c-Myb, a-Myb, b-Myb, and v-Myb,or a gene in the Myc or Myb gene pathway, such as cyclin D1, cyclin D2,cyclin E, CDK4, cdc25A, CDK2, or CDK4.

In one example, an iRNA-lipophilic conjugate that targets a geneexpressed in the nervous system, such as in the brain, e.g., a G72 orD-amino acid oxidase (DAAO) gene, can be delivered to a culturedneuronal cell line, such as an hNT cell line.

In another example, an iRNA-lipophilic conjugate can target a geneencoding a gene in the telomerase pathway, such as TERT or TR/TERC. AniRNA-lipophilic conjugate that targets a gene in the telomerase pathwaycan be delivered, for example, to a human keratinocyte cell line, suchas a HEK cell line, e.g., HEKn or HEKa.

In another example, an iRNA-lipophilic conjugate delivered to atissue-specific cell-line, such as a HEK (keratinocyte), HuVEC(endothelial), 3T3 (fibroblast), or NHDF (fibroblast) cell line, cantarget a gene encoding BCL-2, or VEGF or a VEGF receptor (e.g., VEGFr1,VEGFr2, or VEGFr3).

An iRNA-lipophilic conjugate can be delivered to a subgroup of cellsderived from a particular tissue. For example, an iRNA-lipophilicconjugate can be delivered to a proximal tubular kidney cell line, suchas the mouse cell line mIMCD-3. An iRNA-lipophilic conjugate thattargets a TGF-beta nucleic acid or TGF-beta receptor nucleic acid, forexample, can be delivered to a cell line derived from prostate tissue,e.g., a PC3 or RWPE prostate cell line. An iRNA-lipophilic conjugatedelivered to a prostate tissue cell line can alternatively target apolycomb group gene, such as EZH2.

In another example, an iRNA-lipophilic conjugate can be delivered topancreatic islet b-cells, where for example, it targets a gastricinhibitory polypeptide (GIP) gene, or a GIP-receptor gene.

The iRNA-lipophilic conjugates described herein are not limited in thecell lines to which they can be applied or to the nucleic acids to whichthey can target.

iRNA Agent Structure

The monomers described herein can be used to make oligonucleotides whichare useful as iRNA agents, e.g., RNA molecules, (double-stranded;single-stranded) that mediate RNAi, e.g., with respect to an endogenousgene of a subject or to a gene of a pathogen. In most cases the iRNAagent will incorporate monomers described herein together with naturallyoccurring nucleosides or nucleotides or with other modified nucleosidesor nucleotides. The modified monomers can be present at any position inthe iRNA agent, e.g., at the terminii or in the middle region of an iRNAagent or in a duplex region or in an unpaired region. In a preferredembodiment iRNA agent can have any architecture, e.g., architecturedescribed herein. E.g., it can be incorporated into an iRNA agent havingan overhang structure, a hairpin or other single strand structure or atwo-strand structure, as described herein.

An “RNA agent” as used herein, is an unmodified RNA, modified RNA, ornucleoside surrogate, all of which are defined herein (see, e.g., thesection below entitled RNA Agents). While numerous modified RNAs andnucleoside surrogates are described, preferred examples include thosewhich have greater resistance to nuclease degradation than do unmodifiedRNAs. Preferred examples include those which have a 2′ sugarmodification, a modification in a single strand overhang, preferably a3′ single strand overhang, or, particularly if single stranded, a 5′modification which includes one or more phosphate groups or one or moreanalogs of a phosphate group.

An “iRNA agent” as used herein, is an RNA agent which can, or which canbe cleaved into an RNA agent which can, down regulate the expression ofa target gene, preferably an endogenous or pathogen target RNA. Whilenot wishing to be bound by theory, an iRNA agent may act by one or moreof a number of mechanisms, including post-transcriptional cleavage of atarget mRNA sometimes referred to in the art as RNAi, orpre-transcriptional or pre-translational mechanisms. An iRNA agent caninclude a single strand or can include more than one strands, e.g., itcan be a double stranded iRNA agent. For example, a single stranded iRNAagent can be a microRNA. If the iRNA agent is a single strand it isparticularly preferred that it include a 5′ modification which includesone or more phosphate groups or one or more analogs of a phosphategroup.

The RRMS-containing iRNA agent should include a region of sufficienthomology to the target gene, and be of sufficient length in terms ofnucleotides, such that the iRNA agent, or a fragment thereof, canmediate down regulation of the target gene. It is not necessary thatthere be perfect complementarity between the iRNA agent and the target,but the correspondence must be sufficient to enable the iRNA agent, or acleavage product thereof, to direct sequence specific silencing, e.g.,by RNAi cleavage of the target RNA, e.g., mRNA.

Mismatches to the target mRNA sequence, particularly in the antisensestrand of the iRNA agent, are most tolerated in the terminal regions andif present are preferably in a terminal region or regions, e.g., within6, 5, 4, or 3 nucleotides of the 5′ and/or 3′ terminus, most preferablywithin 6, 5, 4, or 3 nucleotides of the 5′ terminus of the sense strandor 3′ terminus of the antisense strand. The sense strand need only besufficiently complementary with the antisense strand to maintain theover all double strand character of the molecule.

As discussed elsewhere herein, an iRNA agent will often be modified orinclude nucleoside surrogates in addition to the ribose replacementmodification subunit (RRMS). Single stranded regions of an iRNA agentwill often be modified or include nucleoside surrogates, e.g., theunpaired region or regions of a hairpin structure, e.g., a region whichlinks two complementary regions, can have modifications or nucleosidesurrogates. Modification to stabilize one or more 3′- or 5′-terminus ofan iRNA agent, e.g., against exonucleases, or to favor the antisensesRNA agent to enter into RISC are also favored. Modifications caninclude C3 (or C6, C7, C12) amino linkers, thiol linkers, carboxyllinkers, non-nucleotidic spacers (C3, C6, C9, C12, abasic, triethyleneglycol, hexaethylene glycol), special biotin or fluorescein reagentsthat come as phosphoramidites and that have another DMT-protectedhydroxyl group, allowing multiple couplings during RNA synthesis.

Although, in mammalian cells, long dsiRNA agents can induce theinterferon response which is frequently deleterious, short ds iRNAagents do not trigger the interferon response, at least not to an extentthat is deleterious to the cell and host. The iRNA agents featured inthe present invention include molecules which are sufficiently shortthat they do not trigger the interferon response in mammalian cells.Thus, the administration of a composition of an iRNA agent (e.g.,formulated as described herein) to a mammalian cell can be used tosilence expression of the ApoB gene while circumventing the interferonresponse. Molecules that are short enough that they do not trigger aninterferon response are termed sRNA agents or shorter iRNA agentsherein. “sRNA agent or shorter iRNA agent” as used herein, refers to aniRNA agent, e.g., a double stranded RNA agent or single strand agent,that is sufficiently short that it does not induce a deleteriousinterferon response in a human cell, e.g., it has a duplexed region ofless than 60 but preferably less than 50, 40, or 30 nucleotide pairs.The sRNA agent, or a cleavage product thereof, can down regulate atarget gene, e.g., by inducing RNAi with respect to a target RNA,preferably an endogenous or pathogen target RNA.

In addition to homology to target RNA and the ability to down regulate atarget gene, an iRNA agent will preferably have one or more of thefollowing properties:

-   -   (1) it will be of the Formula 1, 2, 3, or 4 set out in the RNA        Agent section below;    -   (2) if single stranded it will have a 5′ modification which        includes one or more phosphate groups or one or more analogs of        a phosphate group;    -   (3) it will, despite modifications, even to a very large number,        or all of the nucleosides, have an antisense strand that can        present bases (or modified bases) in the proper three        dimensional framework so as to be able to form correct base        pairing and form a duplex structure with a homologous target RNA        which is sufficient to allow down regulation of the target,        e.g., by cleavage of the target RNA;    -   (4) it will, despite modifications, even to a very large number,        or all of the nucleosides, still have “RNA-like” properties,        i.e., it will possess the overall structural, chemical and        physical properties of an RNA molecule, even though not        exclusively, or even partly, of ribonucleotide-based content.        For example, an iRNA agent can contain, e.g., a sense and/or an        antisense strand in which all of the nucleotide sugars contain        e.g., 2′ fluoro in place of 2′ hydroxyl. This        deoxyribonucleotide-containing agent can still be expected to        exhibit RNA-like properties. While not wishing to be bound by        theory, the electronegative fluorine prefers an axial        orientation when attached to the C2′ position of ribose. This        spatial preference of fluorine can, in turn, force the sugars to        adopt a C_(3′)-endo pucker. This is the same puckering mode as        observed in RNA molecules and gives rise to the        RNA-characteristic A-family-type helix. Further, since fluorine        is a good hydrogen bond acceptor, it can participate in the same        hydrogen bonding interactions with water molecules that are        known to stabilize RNA structures. (Generally, it is preferred        that a modified moiety at the 2′ sugar position will be able to        enter into H-bonding which is more characteristic of the OH        moiety of a ribonucleotide than the H moiety of a        deoxyribonucleotide. A preferred iRNA agent will: exhibit a        C_(3′)-endo pucker in all, or at least 50, 75, 80, 85, 90, or        95% of its sugars; exhibit a C_(3′)-endo pucker in a sufficient        amount of its sugars that it can give rise to a the        RNA-characteristic A-family-type helix; will have no more than        20, 10, 5, 4, 3, 2, or 1 sugar which is not a C_(3′)-endo pucker        structure. These limitations are particularly preferably in the        antisense strand;    -   (5) regardless of the nature of the modification, and even        though the RNA agent can contain deoxynucleotides or modified        deoxynucleotides, particularly in overhang or other single        strand regions, it is preferred that DNA molecules, or any        molecule in which more than 50, 60, or 70% of the nucleotides in        the molecule, or more than 50, 60, or 70% of the nucleotides in        a duplexed region are deoxyribonucleotides, or modified        deoxyribonucleotides which are deoxy at the 2′ position, are        excluded from the definition of RNA agent.

A “single strand iRNA agent” as used herein, is an iRNA agent which ismade up of a single molecule. It may include a duplexed region, formedby intra-strand pairing, e.g., it may be, or include, a hairpin orpan-handle structure. Single strand iRNA agents are preferably antisensewith regard to the target molecule. In preferred embodiments singlestrand iRNA agents are 5′ phosphorylated or include a phosphoryl analogat the 5′ prime terminus. 5′-phosphate modifications include those whichare compatible with RISC mediated gene silencing. Suitable modificationsinclude: 5′-monophosphate ((HO)2(O)P—O-5′); 5′-diphosphate((HO)2(O)P—O—P(HO)(O)—O-5′); 5′-triphosphate((HO)2(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′); 5′-guanosine cap (7-methylatedor non-methylated) (7m-G-O-5′-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′);5′-adenosine cap (Appp), and any modified or unmodified nucleotide capstructure (N—O-5′-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′);5′-monothiophosphate (phosphorothioate; (HO)2(S)P—O-5′);5′-monodithiophosphate (phosphorodithioate; (HO)(HS)(S)P—O-5′),5′-phosphorothiolate ((HO)2(O)P—S-5′); any additional combination ofoxygen/sulfur replaced monophosphate, diphosphate and triphosphates(e.g. 5′-alpha-thiotriphosphate, 5′-gamma-thiotriphosphate, etc.),5′-phosphoramidates ((HO)2(O)P—NH-5′, (HO)(NH2)(O)P—O-5′),5′-alkylphosphonates (R=alkyl=methyl, ethyl, isopropyl, propyl, etc.,e.g. RP(OH)(O)—O-5′-, (OH)2(O)P-5′-CH2-), 5′-alkyletherphosphonates(R=alkylether=methoxymethyl (MeOCH2-), ethoxymethyl, etc., e.g.RP(OH)(O)—O-5′-). (These modifications can also be used with theantisense strand of a double stranded iRNA.)

A “ds iRNA agent” (abbreviation for “double stranded iRNA agent”) asused herein, is an iRNA agent which includes more than one, andpreferably two, strands in which interchain hybridization can form aregion of duplex structure.

The antisense strand of a double stranded iRNA agent should be equal toor at least, 14, 15, 16 17, 18, 19, 25, 29, 40, or 50 nucleotides inlength. It should be equal to or less than 60, 50, 40, or 30,nucleotides in length. Preferred ranges are 15 to 30, 17 to 25, 19 to23, and 19 to 21 nucleotides in length.

The sense strand of a double stranded iRNA agent should be equal to orat least 14, 15, 16 17, 18, 19, 25, 29, 40, or 50 nucleotides in length.It should be equal to or less than 60, 50, 40, or 30, nucleotides inlength. Preferred ranges are 15 to 30, 17 to 25, 19 to 23, and 19 to 21nucleotides in length.

The double strand portion of a double stranded iRNA agent should beequal to or at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 29, 40,or 50 nucleotide pairs in length. It should be equal to or less than 60,50, 40, or 30, nucleotides pairs in length. Preferred ranges are 15 to30, 17 to 25, 19 to 23, and 19 to 21 nucleotides pairs in length.

It may be desirable to modify one or both of the antisense and sensestrands of a double strand iRNA agent. In some cases they will have thesame modification or the same class of modification but in other casesthe sense and antisense strand will have different modifications, e.g.,in some cases it is desirable to modify only the sense strand. It may bedesirable to modify only the sense strand, e.g., to inactivate it, e.g.,the sense strand can be modified in order to inactivate the sense strandand prevent formation of an active sRNA/protein or RISC. This can beaccomplished by a modification which prevents 5′-phosphorylation of thesense strand, e.g., by modification with a 5′-O-methyl ribonucleotide(see Nykänen et al., (2001) ATP requirements and small interfering RNAstructure in the RNA interference pathway. Cell 107, 309-321.) Othermodifications which prevent phosphorylation can also be used, e.g.,simply substituting the 5′-OH by H rather than O-Me. Alternatively, alarge bulky group may be added to the 5′-phosphate turning it into aphosphodiester linkage, though this may be less desirable asphosphodiesterases can cleave such a linkage and release a functionalsRNA 5′-end. Antisense strand modifications include 5′ phosphorylationas well as any of the other 5′ modifications discussed herein,particularly the 5′ modifications discussed above in the section onsingle stranded iRNA molecules.

It is preferred that the sense and antisense strands be chosen such thatthe ds iRNA agent includes a single strand or unpaired region at one orboth ends of the molecule. Thus, a ds iRNA agent contains sense andantisense strands, preferable paired to contain an overhang, e.g., oneor two 5′ or 3′ overhangs but preferably a 3′ overhang of 2-3nucleotides. Most embodiments will have a 3′ overhang. Preferred sRNAagents will have single-stranded overhangs, preferably 3′ overhangs, of1 to 4, or preferably 2 or 3 nucleotides in length at each end. Theoverhangs can be the result of one strand being longer than the other,or the result of two strands of the same length being staggered. 5′ endsare preferably phosphorylated.

Preferred lengths for the duplexed region is between 15 and 30, mostpreferably 18, 19, 20, 21, 22, and 23 nucleotides in length, e.g., inthe sRNA agent range discussed above. sRNA agents can resemble in lengthand structure the natural Dicer processed products from long dsRNAs.Embodiments in which the two strands of the sRNA agent are linked, e.g.,covalently linked are also included. Hairpin, or other single strandstructures which provide the required double stranded region, andpreferably a 3′ overhang are also within the invention.

As used herein, the phrase “mediates RNAi” refers to the ability of anagent to silence, in a sequence specific manner, a target gene.“Silencing a target gene” means the process whereby a cell containingand/or secreting a certain product of the target gene when not incontact with the agent, will contain and/or secret at least 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, or 90% less of such gene product whencontacted with the agent, as compared to a similar cell which has notbeen contacted with the agent. Such product of the target gene can, forexample, be a messenger RNA (mRNA), a protein, or a regulatory element.While not wishing to be bound by theory, it is believed that silencingby the agents described herein uses the RNAi machinery or process and aguide RNA, e.g., an siRNA agent of 15 to 30 nucleotide pairs.

As used herein, the term “complementary” is used to indicate asufficient degree of complementarity such that stable and specificbinding occurs between a compound of the invention and a target RNAmolecule. Specific binding requires a sufficient degree ofcomplementarity to avoid non-specific binding of the oligomeric compoundto non-target sequences under conditions in which specific binding isdesired, i.e., under physiological conditions in the case of in vivoassays or therapeutic treatment, or in the case of in vitro assays,under conditions in which the assays are performed. The non-targetsequences typically differ by at least 4 nucleotides.

As used herein, an iRNA agent is “sufficiently complementary” to atarget RNA, e.g., a target mRNA, if the iRNA agent reduces theproduction of protein encoded by the target RNA in a cell. The iRNAagent may also be “exactly complementary” (excluding the RRMS containingsubunit(s)) to a target RNA, e.g., the target RNA and the iRNA agentanneal, preferably to form a hybrid made exclusively of Watson-Crickbasepairs in the region of exact complementarity. A “sufficientlycomplementary” target RNA can include an internal region (e.g., of atleast 10 nucleotides) that is exactly complementary to a target RNA.Moreover, in some embodiments, the iRNA agent specifically discriminatesa single-nucleotide difference. In this case, the iRNA agent onlymediates RNAi if exact complementary is found in the region (e.g.,within 7 nucleotides of) the single-nucleotide difference.

RNA agents discussed herein include otherwise unmodified RNA as well asRNA which have been modified, e.g., to improve efficacy, and polymers ofnucleoside surrogates. Unmodified RNA refers to a molecule in which thecomponents of the nucleic acid, namely sugars, bases, and phosphatemoieties, are the same or essentially the same as that which occur innature, preferably as occur naturally in the human body. The art hasreferred to rare or unusual, but naturally occurring, RNAs as modifiedRNAs, see, e.g., Limbach et al., (1994) Summary: the modifiednucleosides of RNA, Nucleic Acids Res. 22: 2183-2196. Such rare orunusual RNAs, often termed modified RNAs (apparently because the aretypically the result of a post transcriptionally modification) arewithin the term unmodified RNA, as used herein. Modified RNA as usedherein refers to a molecule in which one or more of the components ofthe nucleic acid, namely sugars, bases, and phosphate moieties, aredifferent from that which occur in nature, preferably different fromthat which occurs in the human body. While they are referred to asmodified “RNAs,” they will of course, because of the modification,include molecules which are not RNAs. Nucleoside surrogates aremolecules in which the ribophosphate backbone is replaced with anon-ribophosphate construct that allows the bases to the presented inthe correct spatial relationship such that hybridization issubstantially similar to what is seen with a ribophosphate backbone,e.g., non-charged mimics of the ribophosphate backbone. Examples of allof the above are discussed herein.

Much of the discussion below refers to single strand molecules. In manyembodiments of the invention a ds iRNA agent, e.g., a partially doublestranded iRNA agent, is required or preferred. Thus, it is understoodthat double stranded structures (e.g. where two separate molecules arecontacted to form the double stranded region or where the doublestranded region is formed by intramolecular pairing (e.g., a hairpinstructure)) made of the single stranded structures described below arewithin the invention. Preferred lengths are described elsewhere herein.

As nucleic acids are polymers of subunits or monomers, many of themodifications described below occur at a position which is repeatedwithin a nucleic acid, e.g., a modification of a base, or a phosphatemoiety, or the a non-linking O of a phosphate moiety. In some cases themodification will occur at all of the subject positions in the nucleicacid but in many, and infact in most cases it will not. By way ofexample, a modification may only occur at a 3′ or 5′ terminal position,may only occur in a terminal regions, e.g. at a position on a terminalnucleotide or in the last 2, 3, 4, 5, or 10 nucleotides of a strand. Amodification may occur in a double strand region, a single strandregion, or in both. A modification may occur only in the double strandregion of an RNA or may only occur in a single strand region of an RNA.E.g., a phosphorothioate modification at a non-linking O position mayonly occur at one or both termini, may only occur in a terminal regions,e.g., at a position on a terminal nucleotide or in the last 2, 3, 4, 5,or 10 nucleotides of a strand, or may occur in double strand and singlestrand regions, particularly at termini. Similarly, a modification mayoccur on the sense strand, antisense strand, or both. In some cases, thesense and antisense strand will have the same modifications or the sameclass of modifications, but in other cases the sense and antisensestrand will have different modifications, e.g., in some cases it may bedesirable to modify only one strand, e.g. the sense strand.

In some embodiments it is particularly preferred, e.g., to enhancestability, to include particular bases in overhangs, or to includemodified nucleotides or nucleotide surrogates, in single strandoverhangs, e.g., in a 5′ or 3′ overhang, or in both. E.g., it can bedesirable to include purine nucleotides in overhangs. In someembodiments all or some of the bases in a 3′ or 5′ overhang will bemodified, e.g., with a modification described herein. Modifications caninclude, e.g., the use of modifications at the 2′ OH group of the ribosesugar, e.g., the use of deoxyribonucleotides, e.g., deoxythymidine,instead of ribonucleotides, and modifications in the phosphate group,e.g., phosphothioate modifications. Overhangs need not be homologouswith the target sequence.

Modifications and nucleotide surrogates are discussed below.

The scaffold presented above in Formula 1 represents a portion of aribonucleic acid. The basic components are the ribose sugar, the base,the terminal phosphates, and phosphate internucleotide linkers. Wherethe bases are naturally occurring bases, e.g., adenine, uracil, guanineor cytosine, the sugars are the unmodified 2′ hydroxyl ribose sugar (asdepicted) and W, X, Y, and Z are all O, Formula 1 represents a naturallyoccurring unmodified oligoribonucleotide.

Unmodified oligoribonucleotides may be less than optimal in someapplications, e.g., unmodified oligoribonucleotides can be prone todegradation by e.g., cellular nucleases. Nucleases can hydrolyze nucleicacid phosphodiester bonds. However, chemical modifications to one ormore of the above RNA components can confer improved properties, and,e.g., can render oligoribonucleotides more stable to nucleases.Umodified oligoribonucleotides may also be less than optimal in terms ofoffering tethering points for attaching ligands or other moieties to aniRNA agent.

Modified nucleic acids and nucleotide surrogates can include one or moreof:

(i) alteration, e.g., replacement, of one or both of the non-linking (Xand Y) phosphate oxygens and/or of one or more of the linking (W and Z)phosphate oxygens (When the phosphate is in the terminal position, oneof the positions W or Z will not link the phosphate to an additionalelement in a naturally occurring ribonucleic acid. However, forsimplicity of terminology, except where otherwise noted, the W positionat the 5′ end of a nucleic acid and the terminal Z position at the 3′end of a nucleic acid, are within the term “linking phosphate oxygens”as used herein.);

(ii) alteration, e.g., replacement, of a constituent of the ribosesugar, e.g., of the 2′ hydroxyl on the ribose sugar, or wholesalereplacement of the ribose sugar with a structure other than ribose,e.g., as described herein;

(iii) wholesale replacement of the phosphate moiety (bracket I) with“dephospho” linkers;

(iv) modification or replacement of a naturally occurring base;

(v) replacement or modification of the ribose-phosphate backbone(bracket II);

(vi) modification of the 3′ end or 5′ end of the RNA, e.g., removal,modification or replacement of a terminal phosphate group or conjugationof a moiety, e.g. a fluorescently labeled moiety, to either the 3′ or 5′end of RNA.

The terms replacement, modification, alteration, and the like, as usedin this context, do not imply any process limitation, e.g., modificationdoes not mean that one must start with a reference or naturallyoccurring ribonucleic acid and modify it to produce a modifiedribonucleic acid bur rather modified simply indicates a difference froma naturally occurring molecule.

It is understood that the actual electronic structure of some chemicalentities cannot be adequately represented by only one canonical form(i.e. Lewis structure). While not wishing to be bound by theory, theactual structure can instead be some hybrid or weighted average of twoor more canonical forms, known collectively as resonance forms orstructures. Resonance structures are not discrete chemical entities andexist only on paper. They differ from one another only in the placementor “localization” of the bonding and nonbonding electrons for aparticular chemical entity. It can be possible for one resonancestructure to contribute to a greater extent to the hybrid than theothers. Thus, the written and graphical descriptions of the embodimentsof the present invention are made in terms of what the art recognizes asthe predominant resonance form for a particular species. For example,any phosphoroamidate (replacement of a nonlinking oxygen with nitrogen)would be represented by X=O and Y=N in the above figure.

Specific modifications are discussed in more detail below.

The Phosphate Group

The phosphate group is a negatively charged species. The charge isdistributed equally over the two non-linking oxygen atoms (i.e., X and Yin Formula 1 above). However, the phosphate group can be modified byreplacing one of the oxygens with a different substituent. One result ofthis modification to RNA phosphate backbones can be increased resistanceof the oligoribonucleotide to nucleolytic breakdown. Thus while notwishing to be bound by theory, it can be desirable in some embodimentsto introduce alterations which result in either an uncharged linker or acharged linker with unsymmetrical charge distribution.

Examples of modified phosphate groups include phosphorothioate,phosphoroselenates, borano phosphates, borano phosphate esters, hydrogenphosphonates, phosphoroamidates, alkyl or aryl phosphonates andphosphotriesters. Phosphorodithioates have both non-linking oxygensreplaced by sulfur. Unlike the situation where only one of X or Y isaltered, the phosphorus center in the phosphorodithioates is achiralwhich precludes the formation of oligoribonucleotides diastereomers.Diastereomer formation can result in a preparation in which theindividual diastereomers exhibit varying resistance to nucleases.Further, the hybridization affinity of RNA containing chiral phosphategroups can be lower relative to the corresponding unmodified RNAspecies. Thus, while not wishing to be bound by theory, modifications toboth X and Y which eliminate the chiral center, e.g. phosphorodithioateformation, may be desirable in that they cannot produce diastereomermixtures. Thus, X can be any one of S, Se, B, C, H, N, or OR (R is alkylor aryl). Thus Y can be any one of S, Se, B, C, H, N, or OR (R is alkylor aryl). Replacement of X and/or Y with sulfur is preferred.

The phosphate linker can also be modified by replacement of a linkingoxygen (i.e., W or Z in Formula 1) with nitrogen (bridgedphosphoroamidates), sulfur (bridged phosphorothioates) and carbon(bridged methylenephosphonates). The replacement can occur at a terminaloxygen (position W (3′) or position Z (5′). Replacement of W with carbonor Z with nitrogen is preferred.

Candidate agents can be evaluated for suitability as described below.

The Sugar Group

A modified RNA can include modification of all or some of the sugargroups of the ribonucleic acid. E.g., the 2′ hydroxyl group (OH) can bemodified or replaced with a number of different “oxy” or “deoxy”substituents. While not being bound by theory, enhanced stability isexpected since the hydroxyl can no longer be deprotonated to form a 2′alkoxide ion. The 2′ alkoxide can catalyze degradation by intramolecularnucleophilic attack on the linker phosphorus atom. Again, while notwishing to be bound by theory, it can be desirable to some embodimentsto introduce alterations in which alkoxide formation at the 2′ positionis not possible.

Examples of “oxy”-2′ hydroxyl group modifications include alkoxy oraryloxy (OR, e.g., R=H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl orsugar); polyethyleneglycols (PEG), O(CH₂CH₂O)_(n)CH₂CH₂OR; “locked”nucleic acids (LNA) in which the 2′ hydroxyl is connected, e.g., by amethylene bridge, to the 4′ carbon of the same ribose sugar; O-AMINE(AMINE=NH₂; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroaryl amino, or diheteroaryl amino, ethylene diamine,polyamino) and aminoalkoxy, O(CH₂)_(n)AMINE, (e.g., AMINE=NH₂;alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino,heteroaryl amino, or diheteroaryl amino, ethylene diamine, polyamino).It is noteworthy that oligonucleotides containing only the methoxyethylgroup (MOE), (OCH₂CH₂OCH₃, a PEG derivative), exhibit nucleasestabilities comparable to those modified with the robustphosphorothioate modification.

“Deoxy” modifications include hydrogen (i.e. deoxyribose sugars, whichare of particular relevance to the overhang portions of partially dsRNA); halo (e.g., fluoro); amino (e.g. NH₂; alkylamino, dialkylamino,heterocyclyl, arylamino, diaryl amino, heteroaryl amino, diheteroarylamino, or amino acid); NH(CH₂CH₂NH)_(n)CH₂CH₂-AMINE (AMINE=NH₂;alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino,heteroaryl amino, or diheteroaryl amino), —NHC(O)R (R=alkyl, cycloalkyl,aryl, aralkyl, heteroaryl or sugar), cyano; mercapto; alkyl-thio-alkyl;thioalkoxy; and alkyl, cycloalkyl, aryl, alkenyl and alkynyl, which maybe optionally substituted with e.g., an amino functionality. Preferredsubstitutents are 2′-methoxyethyl, 2′-OCH3, 2′-O-allyl, 2′-C-allyl, and2′-fluoro.

The sugar group can also contain one or more carbons that possess theopposite stereochemical configuration than that of the correspondingcarbon in ribose. Thus, a modified RNA can include nucleotidescontaining e.g., arabinose, as the sugar.

Modified RNAs can also include “abasic” sugars, which lack a nucleobaseat C-1′. These abasic sugars can also be further contain modificationsat one or more of the constituent sugar atoms.

To maximize nuclease resistance, the 2′ modifications can be used incombination with one or more phosphate linker modifications (e.g.,phosphorothioate). The so-called “chimeric” oligonucleotides are thosethat contain two or more different modifications.

The modification can also entail the wholesale replacement of a ribosestructure with another entity at one or more sites in the iRNA agent.These modifications are described in section entitled RiboseReplacements for RRMSs.

Candidate modifications can be evaluated as described below.

Replacement of the Phosphate Group

The phosphate group can be replaced by non-phosphorus containingconnectors (cf. Bracket I in Formula 1 above). While not wishing to bebound by theory, it is believed that since the charged phosphodiestergroup is the reaction center in nucleolytic degradation, its replacementwith neutral structural mimics should impart enhanced nucleasestability. Again, while not wishing to be bound by theory, it can bedesirable, in some embodiment, to introduce alterations in which thecharged phosphate group is replaced by a neutral moiety.

Examples of moieties which can replace the phosphate group includesiloxane, carbonate, carboxymethyl, carbamate, amide, thioether,ethylene oxide linker, sulfonate, sulfonamide, thioformacetal,formacetal, oxime, methyleneimino, methylenemethylimino,methylenehydrazo, methylenedimethylhydrazo and methyleneoxymethylimino.Preferred replacements include the methylenecarbonylamino andmethylenemethylimino groups.

Candidate modifications can be evaluated as described below.

Replacement of Ribophosphate Backbone

Oligonucleotide-mimicking scaffolds can also be constructed wherein thephosphate linker and ribose sugar are replaced by nuclease resistantnucleoside or nucleotide surrogates (see Bracket II of Formula 1 above).While not wishing to be bound by theory, it is believed that the absenceof a repetitively charged backbone diminishes binding to proteins thatrecognize polyanions (e.g. nucleases). Again, while not wishing to bebound by theory, it can be desirable in some embodiment, to introducealterations in which the bases are tethered by a neutral surrogatebackbone.

Examples include the mophilino, cyclobutyl, pyrrolidine and peptidenucleic acid (PNA) nucleoside surrogates. A preferred surrogate is a PNAsurrogate.

Candidate modifications can be evaluated as described below.

Terminal Modifications

The 3′ and 5′ ends of an oligonucleotide can be modified. Suchmodifications can be at the 3′ end, 5′ end or both ends of the molecule.They can include modification or replacement of an entire terminalphosphate or of one or more of the atoms of the phosphate group. E.g.,the 3′ and 5′ ends of an oligonucleotide can be conjugated to otherfunctional molecular entities such as labeling moieties, e.g.,fluorophores (e.g., pyrene, TAMRA, fluorescein, Cy3 or Cy5 dyes) orprotecting groups (based e.g., on sulfur, silicon, boron or ester). Thefunctional molecular entities can be attached to the sugar through aphosphate group and/or a spacer. The terminal atom of the spacer canconnect to or replace the linking atom of the phosphate group or theC-3′ or C-5′ O, N, S or C group of the sugar. Alternatively, the spacercan connect to or replace the terminal atom of a nucleotide surrogate(e.g., PNAs). These spacers or linkers can include e.g., —(CH₂)_(n)—,—(CH₂)_(n)N—, —(CH₂)_(n)O—, —(CH₂)_(n)S—, O(CH₂CH₂O)_(n)CH₂CH₂OH (e.g.,n=3 or 6), abasic sugars, amide, carboxy, amine, oxyamine, oxyimine,thioether, disulfide, thiourea, sulfonamide, or morpholino, or biotinand fluorescein reagents. When a spacer/phosphate-functional molecularentity-spacer/phosphate array is interposed between two strands of iRNAagents, this array can substitute for a hairpin RNA loop in ahairpin-type RNA agent. The 3′ end can be an —OH group. While notwishing to be bound by theory, it is believed that conjugation ofcertain moieties can improve transport, hybridization, and specificityproperties. Again, while not wishing to be bound by theory, it may bedesirable to introduce terminal alterations that improve nucleaseresistance. Other examples of terminal modifications include dyes,intercalating agents (e.g. acridines), cross-linkers (e.g. psoralene,mitomycin C), porphyrins (TPPC4, texaphyrin, Sapphyrin), polycyclicaromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificialendonucleases (e.g. EDTA), lipophilic carriers (e.g., cholesterol,cholic acid, adamantane acetic acid, 1-pyrene butyric acid,dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexylgroup, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecylgroup, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid,O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine) and peptideconjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents,phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]₂,polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes,haptens (e.g. biotin), transport/absorption facilitators (e.g., aspirin,vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole,bisimidazole, histamine, imidazole clusters, acridine-imidazoleconjugates, Eu3+complexes of tetraazamacrocycles).

Terminal modifications can be added for a number of reasons, includingas discussed elsewhere herein to modulate activity or to modulateresistance to degradation. Terminal modifications useful for modulatingactivity include modification of the 5′ end with phosphate or phosphateanalogs. E.g., in preferred embodiments iRNA agents, especiallyantisense strands, are 5′ phosphorylated or include a phosphoryl analogat the 5′ prime terminus. 5′-phosphate modifications include those whichare compatible with RISC mediated gene silencing. Suitable modificationsinclude: 5′-monophosphate ((HO)2(O)P—O-5′); 5′-diphosphate((HO)2(O)P—O—P(HO)(O)—O-5′); 5′-triphosphate((HO)2(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′); 5′-guanosine cap (7-methylatedor non-methylated) (7m-G-O-5′-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′);5′-adenosine cap (Appp), and any modified or unmodified nucleotide capstructure (N—O-5′-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′);5′-monothiophosphate (phosphorothioate; (HO)2(S)P—O-5′);5′-monodithiophosphate (phosphorodithioate; (HO)(HS)(S)P—O-5′),5′-phosphorothiolate ((HO)2(O)P—S-5′); any additional combination ofoxgen/sulfur replaced monophosphate, diphosphate and triphosphates (e.g.5′-alpha-thiotriphosphate, 5′-gamma-thiotriphosphate, etc.),5′-phosphoramidates ((HO)2(O)P—NH-5′, (HO)(NH2)(O)P—O-5′),5′-alkylphosphonates (R=alkyl=methyl, ethyl, isopropyl, propyl, etc.,e.g. RP(OH)(O)—O-5′-, (OH)2(O)P-5′-CH2-), 5′-alkyletherphosphonates(R=alkylether=methoxymethyl (MeOCH2-), ethoxymethyl, etc., e.g.RP(OH)(O)—O-5′-).

Terminal modifications can also be useful for monitoring distribution,and in such cases the preferred groups to be added include fluorophores,e.g., fluorscein or an Alexa dye, e.g., Alexa 488. Terminalmodifications can also be useful for enhancing uptake, usefulmodifications for this include cholesterol. Terminal modifications canalso be useful for cross-linking an RNA agent to another moiety;modifications useful for this include mitomycin C.

Candidate modifications can be evaluated as described below.

The Bases

Adenine, guanine, cytosine and uracil are the most common bases found inRNA. These bases can be modified or replaced to provide RNA's havingimproved properties. E.g., nuclease resistant oligoribonucleotides canbe prepared with these bases or with synthetic and natural nucleobases(e.g., inosine, thymine, xanthine, hypoxanthine, nubularine,isoguanisine, or tubercidine) and any one of the above modifications.Alternatively, substituted or modified analogs of any of the abovebases, e.g., “unusual bases” and “universal bases” described herein, canbe employed. Examples include without limitation 2-aminoadenine,6-methyl and other alkyl derivatives of adenine and guanine, 2-propyland other alkyl derivatives of adenine and guanine, 5-halouracil andcytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine andthymine, 5-uracil (pseudouracil), 4-thiouracil, 5-halouracil,5-(2-aminopropyl)uracil, 5-amino allyl uracil, 8-halo, amino, thiol,thioalkyl, hydroxyl and other 8-substituted adenines and guanines,5-trifluoromethyl and other 5-substituted uracils and cytosines,7-methylguanine, 5-substituted pyrimidines, 6-azapyrimidines and N-2,N-6 and O-6 substituted purines, including 2-aminopropyladenine,5-propynyluracil and 5-propynylcytosine, dihydrouracil,3-deaza-5-azacytosine, 2-aminopurine, 5-alkyluracil, 7-alkylguanine,5-alkyl cytosine, 7-deazaadenine, N6, N6-dimethyladenine,2,6-diaminopurine, 5-amino-allyl-uracil, N3-methyluracil, substituted1,2,4-triazoles, 2-pyridinone, 5-nitroindole, 3-nitropyrrole,5-methoxyuracil, uracil-5-oxyacetic acid, 5-methoxycarbonylmethyluracil,5-methyl-2-thiouracil, 5-methoxycarbonylmethyl-2-thiouracil,5-methylaminomethyl-2-thiouracil, 3-(3-amino-3carboxypropyl)uracil,3-methylcytosine, 5-methylcytosine, N⁴-acetyl cytosine, 2-thiocytosine,N6-methyladenine, N6-isopentyladenine,2-methylthio-N-6-isopentenyladenine, N-methylguanines, or O-alkylatedbases. Further purines and pyrimidines include those disclosed in U.S.Pat. No. 3,687,808, those disclosed in the Concise Encyclopedia OfPolymer Science And Engineering, pages 858-859, Kroschwitz, J. I., ed.John Wiley & Sons, 1990, and those disclosed by Englisch et al.,Angewandte Chemie, International Edition, 1991, 30, 613.

Generally, base changes are less preferred for promoting stability, butthey can be useful for other reasons, e.g., some, e.g.,2,6-diaminopurine and 2 amino purine, are fluorescent. Modified basescan reduce target specificity. This should be taken into considerationin the design of iRNA agents.

An iRNA agent can have a ZXY structure, such as is described in co-ownedPCT Application No. PCT/US2004/07070 filed on Mar. 8, 2004.

An iRNA agent can be complexed with an amphipathic moiety. Exemplaryamphipathic moieties for use with iRNA agents are described in co-ownedPCT Application No. PCT/US2004/07070 filed on Mar. 8, 2004.

In another embodiment, the iRNA agent can be complexed to a deliveryagent that features a modular complex. The complex can include a carrieragent linked to one or more of (preferably two or more, more preferablyall three of): (a) a condensing agent (e.g., an agent capable ofattracting, e.g., binding, a nucleic acid, e.g., through ionic orelectrostatic interactions); (b) a fusogenic agent (e.g., an agentcapable of fusing and/or being transported through a cell membrane); and(c) a targeting group, e.g., a cell or tissue targeting agent, e.g., alectin, glycoprotein, lipid or protein, e.g., an antibody, that binds toa specified cell type. iRNA agents complexed to a delivery agent aredescribed in co-owned PCT Application No. PCT/US2004/07070 filed on Mar.8, 2004.

An iRNA agent can have non-canonical pairings, such as between the senseand antisense sequences of the iRNA duplex. Exemplary features ofnon-canonical iRNA agents are described in co-owned PCT Application No.PCT/US2004/07070 filed on Mar. 8, 2004.

Candidate modifications can be evaluated as described below.

Evaluation of Candidate iRNA Agents

A candidate iRNA agent can be evaluated for its ability to downregulatetarget gene expression. For example, a candidate iRNA agent can beprovided, and contacted with a cell that expresses the target geneeither endogenously or because it has been transfected with a constructfrom which the target gene can be expressed. The level of target geneexpression prior to and following contact with the candidate iRNA agentcan be compared, e.g. on an mRNA or protein level. If it is determinedthat the amount of RNA or protein expressed from the target gene islower following contact with the iRNA agent, then it can be concludedthat the iRNA agent down-regulates target gene expression. The level oftarget RNA or protein in the cell can be determined by any methoddesired. For example, the level of target RNA can be determined byNorthern blot analysis, reverse transcription coupled with polymerasechain reaction (RT-PCR), or RNAse protection assay. The level of proteincan be determined, for example, by Western blot analysis.

Stability Testing, Modification, and Retesting of iRNA Agents

A candidate iRNA agent can be evaluated with respect to stability, e.g.,its susceptibility to cleavage by an endonuclease or exonuclease, suchas when the iRNA agent is introduced into the body of a subject. Methodscan be employed to identify sites that are susceptible to modification,particularly cleavage, e.g., cleavage by a component found in the bodyof a subject.

When sites susceptible to cleavage are identified, a further iRNA agentcan be designed and/or synthesized wherein the potential cleavage siteis made resistant to cleavage, e.g. by introduction of a 2′-modificationon the site of cleavage, e.g. a 2′-O-methyl group. This further iRNAagent can be retested for stability, and this process may be iterateduntil an iRNA agent is found exhibiting the desired stability.

In Vivo Testing

An iRNA agent identified as being capable of inhibiting target geneexpression can be tested for functionality in vivo in an animal model(e.g., in a mammal, such as in mouse or rat). For example, the iRNAagent can be administered to an animal, and the iRNA agent evaluatedwith respect to its biodistribution, stability, and its ability toinhibit target gene expression.

The iRNA agent can be administered directly to the target tissue, suchas by injection, or the iRNA agent can be administered to the animalmodel in the same manner that it would be administered to a human.

The iRNA agent can also be evaluated for its intracellular distribution.The evaluation can include determining whether the iRNA agent was takenup into the cell. The evaluation can also include determining thestability (e.g., the half-life) of the iRNA agent. Evaluation of an iRNAagent in vivo can be facilitated by use of an iRNA agent conjugated to atraceable marker (e.g., a fluorescent marker such as fluorescein; aradioactive label, such as ³⁵S, ³²P, ³³P, or ³H; gold particles; orantigen particles for immunohistochemistry).

An iRNA agent useful for monitoring biodistribution can lack genesilencing activity in vivo. For example, the iRNA agent can target agene not present in the animal (e.g., an iRNA agent injected into mousecan target luciferase), or an iRNA agent can have a non-sense sequence,which does not target any gene, e.g., any endogenous gene).Localization/biodistribution of the iRNA can be monitored, e.g. by atraceable label attached to the iRNA agent, such as a traceable agentdescribed above.

The iRNA agent can be evaluated with respect to its ability to downregulate target gene expression. Levels of target gene expression invivo can be measured, for example, by in situ hybridization, or by theisolation of RNA from tissue prior to and following exposure to the iRNAagent. Where the animal needs to be sacrificed in order to harvest thetissue, an untreated control animal will serve for comparison. TargetmRNA can be detected by any desired method, including but not limited toRT-PCR, Northern blot, branched-DNA assay, or RNAase protection assay.Alternatively, or additionally, target gene expression can be monitoredby performing Western blot analysis on tissue extracts treated with theiRNA agent.

REFERENCES

General References

The oligoribonucleotides and oligoribonucleosides used in accordancewith this invention may be with solid phase synthesis, see for example“Oligonucleotide synthesis, a practical approach”, Ed. M. J. Gait, IRLPress, 1984; “Oligonucleotides and Analogues, A Practical Approach”, Ed.F. Eckstein, IRL Press, 1991 (especially Chapter 1, Modern machine-aidedmethods of oligodeoxyribonucleotide synthesis, Chapter 2,Oligoribonucleotide synthesis, Chapter3,2′-O-Methyloligoribonucleotide-s: synthesis and applications, Chapter4, Phosphorothioate oligonucleotides, Chapter 5, Synthesis ofoligonucleotide phosphorodithioates, Chapter 6, Synthesis ofoligo-2′-deoxyribonucleoside methylphosphonates, and. Chapter 7,Oligodeoxynucleotides containing modified bases. Other particularlyuseful synthetic procedures, reagents, blocking groups and reactionconditions are described in Martin, P., Helv. Chim. Acta, 1995, 78,486-504; Beaucage, S. L. and Iyer, R. P., Tetrahedron, 1992, 48,2223-2311 and Beaucage, S. L. and Iyer, R. P., Tetrahedron, 1993, 49,6123-6194, or references referred to therein.

Modification described in WO 00/44895, WO01/75164, or WO02/44321 can beused herein.

The disclosure of all publications, patents, and published patentapplications listed herein are hereby incorporated by reference.

Phosphate Group References

The preparation of phosphinate oligoribonucleotides is described in U.S.Pat. No. 5,508,270. The preparation of alkyl phosphonateoligoribonucleotides is described in U.S. Pat. No. 4,469,863. Thepreparation of phosphoramidite oligoribonucleotides is described in U.S.Pat. No. 5,256,775 or U.S. Pat. No. 5,366,878. The preparation ofphosphotriester oligoribonucleotides is described in U.S. Pat. No.5,023,243. The preparation of borano phosphate oligoribonucleotide isdescribed in U.S. Pat. Nos. 5,130,302 and 5,177,198. The preparation of3′-Deoxy-3′-amino phosphoramidate oligoribonucleotides is described inU.S. Pat. No. 5,476,925. 3′-Deoxy-3′-methylenephosphonateoligoribonucleotides is described in An, H, et al. J. Org. Chem. 2001,66, 2789-2801. Preparation of sulfur bridged nucleotides is described inSproat et al. Nucleosides Nucleotides 1988, 7, 651 and Crosstick et al.Tetrahedron Lett. 1989, 30, 4693.

Sugar Group References

Modifications to the 2′ modifications can be found in Verma, S. et al.Annu. Rev. Biochem. 1998, 67, 99-134 and all references therein.Specific modifications to the ribose can be found in the followingreferences: 2′-fluoro (Kawasaki et. al., J. Med. Chem., 1993, 36,831-841), 2′-MOE (Martin, P. Helv. Chim. Acta 1996, 79, 1930-1938),“LNA” (Wengel, J. Acc. Chem. Res. 1999, 32, 301-310).

Replacement of the Phosphate Group References

Methylenemethylimino linked oligoribonucleosides, also identified hereinas MMI linked oligoribonucleosides, methylenedimethylhydrazo linkedoligoribonucleosides, also identified herein as MDH linkedoligoribonucleosides, and methylenecarbonylamino linkedoligonucleosides, also identified herein as amide-3 linkedoligoribonucleosides, and methyleneaminocarbonyl linkedoligonucleosides, also identified herein as amide-4 linkedoligoribonucleosides as well as mixed backbone compounds having, as forinstance, alternating MMI and PO or PS linkages can be prepared as isdescribed in U.S. Pat. Nos. 5,378,825, 5,386,023, 5,489,677 and inpublished PCT applications PCT/US92/04294 and PCT/US92/04305 (publishedas WO 92/20822 WO and 92/20823, respectively). Formacetal andthioformacetal linked oligoribonucleosides can be prepared as isdescribed in U.S. Pat. Nos. 5,264,562 and 5,264,564. Ethylene oxidelinked oligoribonucleosides can be prepared as is described in U.S. Pat.No. 5,223,618. Siloxane replacements are described in Cormier, J. F. etal. Nucleic Acids Res. 1988, 16, 4583. Carbonate replacements aredescribed in Tittensor, J. R. J. Chem. Soc. C 1971, 1933. Carboxymethylreplacements are described in Edge, M. D. et al. J. Chem. Soc. PerkinTrans. 1 1972, 1991. Carbamate replacements are described in Stirchak,E. P. Nucleic Acids Res. 1989, 17, 6129.

Replacement of the Phosphate-Ribose Backbone References

Cyclobutyl sugar surrogate compounds can be prepared as is described inU.S. Pat. No. 5,359,044. Pyrrolidine sugar surrogate can be prepared asis described in U.S. Pat. No. 5,519,134. Morpholino sugar surrogates canbe prepared as is described in U.S. Pat. Nos. 5,142,047 and 5,235,033,and other related patent disclosures. Peptide Nucleic Acids (PNAs) areknown per se and can be prepared in accordance with any of the variousprocedures referred to in Peptide Nucleic Acids (PNA): Synthesis,Properties and Potential Applications, Bioorganic & Medicinal Chemistry,1996, 4, 5-23. They may also be prepared in accordance with U.S. Pat.No. 5,539,083.

Terminal Modification References

Terminal modifications are described in Manoharan, M. et al. Antisenseand Nucleic Acid Drug Development 12, 103-128 (2002) and referencestherein.

Bases References

N-2 substitued purine nucleoside amidites can be prepared as isdescribed in U.S. Pat. No. 5,459,255. 3-Deaza purine nucleoside amiditescan be prepared as is described in U.S. Pat. No. 5,457,191.5,6-Substituted pyrimidine nucleoside amidites can be prepared as isdescribed in U.S. Pat. No. 5,614,617. 5-Propynyl pyrimidine nucleosideamidites can be prepared as is described in U.S. Pat. No. 5,484,908.Additional references can be disclosed in the above section on basemodifications.

Preferred iRNA Agents

Preferred RNA agents have the following structure (see Formula 2 below):

Referring to Formula 2 above, R¹, R², and R³ are each, independently, H,(i.e. abasic nucleotides), adenine, guanine, cytosine and uracil,inosine, thymine, xanthine, hypoxanthine, nubularine, tubercidine,isoguanisine, 2-aminoadenine, 6-methyl and other alkyl derivatives ofadenine and guanine, 2-propyl and other alkyl derivatives of adenine andguanine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine,6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil),4-thiouracil, 5-halouracil, 5-(2-aminopropyl)uracil, 5-amino allyluracil, 8-halo, amino, thiol, thioalkyl, hydroxyl and other8-substituted adenines and guanines, 5-trifluoromethyl and other5-substituted uracils and cytosines, 7-methylguanine, 5-substitutedpyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines,including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine,dihydrouracil, 3-deaza-5-azacytosine, 2-aminopurine, 5-alkyluracil,7-alkylguanine, 5-alkyl cytosine, 7-deazaadenine, 7-deazaguanine, N6,N6-dimethyladenine, 2,6-diaminopurine, 5-amino-allyl-uracil,N3-methyluracil, substituted 1,2,4-triazoles, 2-pyridinone,5-nitroindole, 3-nitropyrrole, 5-methoxyuracil, uracil-5-oxyacetic acid,5-methoxycarbonylmethyluracil, 5-methyl-2-thiouracil,5-methoxycarbonylmethyl-2-thiouracil, 5-methylaminomethyl-2-thiouracil,3-(3-amino-3carboxypropyl)uracil, 3-methylcytosine, 5-methylcytosine,N⁴-acetyl cytosine, 2-thiocytosine, N6-methyladenine,N6-isopentyladenine, 2-methylthio-N-6-isopentenyladenine,N-methylguanines, or O-alkylated bases.

R⁴, R⁵, and R⁶ are each, independently, OR⁸, O(CH₂CH₂O)_(m)CH₂CH₂OR⁸;O(CH₂)_(n)R⁹; O(CH₂)_(n)OR⁹, H; halo; NH₂; NHR⁸; N(R⁸)₂;NH(CH₂CH₂NH)_(m)CH₂CH₂NHR⁹; NHC(O)R⁸; cyano; mercapto, SR⁸;alkyl-thio-alkyl; alkyl, aralkyl, cycloalkyl, aryl, heteroaryl, alkenyl,alkynyl, each of which may be optionally substituted with halo, hydroxy,oxo, nitro, haloalkyl, alkyl, alkaryl, aryl, aralkyl, alkoxy, aryloxy,amino, alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino,heteroaryl amino, diheteroaryl amino, acylamino, alkylcarbamoyl,arylcarbamoyl, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl,alkanesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido,alkylcarbonyl, acyloxy, cyano, or ureido; or R⁴, R⁵, or R⁶ togethercombine with R⁷ to form an [—O—CH₂—] covalently bound bridge between thesugar 2′ and 4′ carbons.

-   -   H; OH; OCH₃; W¹; an abasic nucleotide; or absent;    -   (a preferred A1, especially with regard to anti-sense strands,        is chosen from 5′-monophosphate ((HO)₂(O)P—O-5′), 5′-diphosphate        ((HO)₂(O)P—O—P(HO)(O)—O-5′), 5′-triphosphate        ((HO)₂(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′), 5′-guanosine cap        (7-methylated or non-methylated)        (7m-G-O-5′-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′), 5′-adenosine        cap (Appp), and any modified or unmodified nucleotide cap        structure (N—O-5′-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′),        5′-monothiophosphate (phosphorothioate; (HO)₂(S)P—O-5′),        5′-monodithiophosphate (phosphorodithioate; (HO)(HS)(S)P—O-5′),        5′-phosphorothiolate ((HO)₂(O)P—S-5′); any additional        combination of oxgen/sulfur replaced monophosphate, diphosphate        and triphosphates (e.g. 5′-alpha-thiotriphosphate,        5′-gamma-thiotriphosphate, etc.), 5′-phosphoramidates        ((HO)₂(O)P—NH-5′, (HO)(NH₂)(O)P—O-5′), 5′-alkylphosphonates        (R=alkyl=methyl, ethyl, isopropyl, propyl, etc., e.g.        RP(OH)(O)—O-5′-, (OH)₂(O)P-5′-CH₂—), 5′-alkyletherphosphonates        (R=alkylether=methoxymethyl (MeOCH₂—), ethoxymethyl, etc., e.g.        RP(OH)(O)—O-5′-)).

-   -   H; Z⁴; an inverted nucleotide; an abasic nucleotide; or absent.

W¹ is OH, (CH₂)_(n)R¹⁰, (CH₂)_(n)NHR¹⁰, (CH₂)_(n)OR¹⁰, (CH₂)_(n)SR¹⁰;O(CH₂)_(n)R¹⁰; O(CH₂)_(n)OR¹⁰, O(CH₂)_(n)NR¹⁰, O(CH₂)_(n)SR¹⁰;O(CH₂)_(n)SS(CH₂)_(n)OR¹⁰, O(CH₂)_(n)C(O)OR¹⁰, NH(CH₂)_(n)R¹⁰;NH(CH₂)_(n)NR¹⁰; NH(CH₂)_(n)OR¹⁰, NH(CH₂)_(n)SR¹⁰; S(CH₂)_(n)R¹⁰,S(CH₂)_(n)NR¹⁰, S(CH₂)_(n)OR¹⁰, S(CH₂)_(n)SR¹⁰O(CH₂CH₂O)_(m)CH₂CH₂OR¹⁰;O(CH₂CH₂O)_(m)CH₂CH₂NHR¹⁰, NH(CH₂CH₂NH)_(m)CH₂CH₂NHR¹⁰; Q-R¹⁰, O-Q-R¹⁰,N-Q-R¹⁰, S-Q-R¹⁰ or —O—. W⁴ is O, CH₂, NH, or S.

X¹, X², X³, and X⁴ are each, independently, O or S.

Y¹, Y², Y³, and Y⁴ are each, independently, OH, O⁻, OR⁸, S, Se, BH₃ ⁻,H, NHR⁹, N(R⁹)₂ alkyl, cycloalkyl, aralkyl, aryl, or heteroaryl, each ofwhich may be optionally substituted.

Z¹, Z², and Z³ are each independently O, CH₂, NH, or S. Z⁴ is OH,(CH₂)_(n)R¹⁰, (CH₂)_(n)NHR¹⁰, (CH₂)_(n)OR¹⁰, (CH₂)_(n)SR¹⁰;O(CH₂)_(n)R¹⁰; O(CH₂)_(n)OR¹⁰, O(CH₂)_(n)NR¹⁰, O(CH₂)_(n)SR¹⁰,O(CH₂)_(n)SS(CH₂)_(n)OR¹⁰, O(CH₂)_(n)C(O)OR¹⁰; NH(CH₂)_(n)R¹⁰;NH(CH₂)_(n)NR¹⁰; NH(CH₂)_(n)OR¹⁰, NH(CH₂)_(n)SR¹⁰; S(CH₂)_(n)R¹⁰,S(CH₂)_(n)NR¹⁰, S(CH₂)_(n)OR¹⁰, S(CH₂)_(n)SR¹⁰ O(CH₂CH₂O)_(m)CH₂CH₂OR¹⁰,O(CH₂CH₂O)_(m)CH₂CH₂NHR¹⁰, NH(CH₂CH₂NH)_(m)CH₂CH₂NHR¹⁰; Q-R¹⁰, O-Q-R¹⁰N-Q-R¹⁰, S-Q-R¹⁰.

x is 5-100, chosen to comply with a length for an RNA agent describedherein.

R⁷ is H; or is together combined with R⁴, R⁵, or R⁶ to form an [—O—CH₂—]covalently bound bridge between the sugar 2′ and 4′ carbons.

R⁸ is alkyl, cycloalkyl, aryl, aralkyl, heterocyclyl, heteroaryl, aminoacid, or sugar; R⁹ is NH₂, alkylamino, dialkylamino, heterocyclyl,arylamino, diaryl amino, heteroaryl amino, diheteroaryl amino, or aminoacid; and R¹⁰ is H; fluorophore (pyrene, TAMRA, fluorescein, Cy3 or Cy5dyes); sulfur, silicon, boron or ester protecting group; intercalatingagents (e.g. acridines), cross-linkers (e.g. psoralene, mitomycin C),porphyrins (TPPC4, texaphyrin, Sapphyrin), polycyclic aromatichydrocarbons (e.g., phenazine, dihydrophenazine), artificialendonucleases (e.g. EDTA), lipohilic carriers (cholesterol, cholic acid,adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone,1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol,borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid,myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid,dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g.,antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino,mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]₂, polyamino; alkyl,cycloalkyl, aryl, aralkyl, heteroaryl; radiolabelled markers, enzymes,haptens (e.g. biotin), transport/absorption facilitators (e.g., aspirin,vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole,bisimidazole, histamine, imidazole clusters, acridine-imidazoleconjugates, Eu3+ complexes of tetraazamacrocycles); or an RNA agent. mis 0-1,000,000, and n is 0-20. Q is a spacer selected from the groupconsisting of abasic sugar, amide, carboxy, oxyamine, oxyimine,thioether, disulfide, thiourea, sulfonamide, or morpholino, biotin orfluorescein reagents.

Preferred RNA agents in which the entire phosphate group has beenreplaced have the following structure (see Formula 3 below):

Referring to Formula 3, A¹⁰-A⁴⁰ is L-G-L; A¹⁰ and/or A⁴⁰ may be absent,in which Lisa linker, wherein one or both L may be present or absent andis selected from the group consisting of CH₂(CH₂)_(g); N(CH₂)_(g);O(CH₂)_(g); S(CH₂)_(g). G is a functional group selected from the groupconsisting of siloxane, carbonate, carboxymethyl, carbamate, amide,thioether, ethylene oxide linker, sulfonate, sulfonamide,thioformacetal, formacetal, oxime, methyleneimino, methylenemethylimino,methylenehydrazo, methylenedimethylhydrazo and methyleneoxymethylimino.

R¹⁰, R²⁰, and R³⁰ are each, independently, H, (i.e. abasic nucleotides),adenine, guanine, cytosine and uracil, inosine, thymine, xanthine,hypoxanthine, nubularine, tubercidine, isoguanisine, 2-aminoadenine,6-methyl and other alkyl derivatives of adenine and guanine, 2-propyland other alkyl derivatives of adenine and guanine, 5-halouracil andcytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine andthymine, 5-uracil (pseudouracil), 4-thiouracil, 5-halouracil,5-(2-aminopropyl)uracil, 5-amino allyl uracil, 8-halo, amino, thiol,thioalkyl, hydroxyl and other 8-substituted adenines and guanines,5-trifluoromethyl and other 5-substituted uracils and cytosines,7-methylguanine, 5-substituted pyrimidines, 6-azapyrimidines and N-2,N-6 and O-6 substituted purines, including 2-aminopropyladenine,5-propynyluracil and 5-propynylcytosine, dihydrouracil,3-deaza-5-azacytosine, 2-aminopurine, 5-alkyluracil, 7-alkylguanine,5-alkyl cytosine, 7-deazaadenine, 7-deazaguanine, N6,N6-dimethyladenine, 2,6-diaminopurine, 5-amino-allyl-uracil,N3-methyluracil substituted 1,2,4-triazoles, 2-pyridinone,5-nitroindole, 3-nitropyrrole, 5-methoxyuracil, uracil-5-oxyacetic acid,5-methoxycarbonylmethyluracil, 5-methyl-2-thiouracil,5-methoxycarbonylmethyl-2-thiouracil, 5-methylaminomethyl-2-thiouracil,3-(3-amino-3carboxypropyl)uracil, 3-methylcytosine, 5-methylcytosine,N⁴-acetyl cytosine, 2-thiocytosine, N6-methyladenine,N6-isopentyladenine, 2-methylthio-N-6-isopentenyladenine,N-methylguanines, or O-alkylated bases.

R⁴⁰, R⁵⁰, and R⁶⁰ are each, independently, OR⁸, O(CH₂CH₂O)_(m)CH₂CH₂OR⁸;O(CH₂)_(n)R⁹; O(CH₂)_(n)OR⁹, H; halo; NH₂; NHR⁸; N(R⁸)₂;NH(CH₂CH₂NH)_(m)CH₂CH₂R⁹; NHC(O)R⁸; cyano; mercapto, SR⁷;alkyl-thio-alkyl; alkyl, aralkyl, cycloalkyl, aryl, heteroaryl, alkenyl,alkynyl, each of which may be optionally substituted with halo, hydroxy,oxo, nitro, haloalkyl, alkyl, alkaryl, aryl, aralkyl, alkoxy, aryloxy,amino, alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino,heteroaryl amino, diheteroaryl amino, acylamino, alkylcarbamoyl,arylcarbamoyl, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl,alkanesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido,alkylcarbonyl, acyloxy, cyano, and ureido groups; or R⁴⁰, R⁵⁰, or R⁶⁰together combine with R⁷⁰ to form an [—O—CH₂—] covalently bound bridgebetween the sugar 2′ and 4′ carbons.

x is 5-100 or chosen to comply with a length for an RNA agent describedherein.

R⁷⁰ is H; or is together combined with R⁴⁰, R⁵⁰, or R⁶⁰ to form an[—O—CH₂—] covalently bound bridge between the sugar 2′ and 4′ carbons.

R⁸ is alkyl, cycloalkyl, aryl, aralkyl, heterocyclyl, heteroaryl, aminoacid, or sugar; and R⁹ is NH₂, alkylamino, dialkylamino, heterocyclyl,arylamino, diaryl amino, heteroaryl amino, diheteroaryl amino, or aminoacid. m is 0-1,000,000, n is 0-20, and g is 0-2.

Preferred nucleoside surrogates have the following structure (seeFormula 4 below):SLR¹⁰⁰-(M-SLR²⁰⁰)_(x)-M-SLR³⁰⁰   Formula 4

S is a nucleoside surrogate selected from the group consisting ofmophilino, cyclobutyl, pyrrolidine and peptide nucleic acid. L is alinker and is selected from the group consisting of CH₂(CH₂)_(g);N(CH₂)_(g); O(CH₂)_(g); S(CH₂)_(g); —C(O)(CH₂)_(n)— or may be absent. Mis an amide bond; sulfonamide; sulfinate; phosphate group; modifiedphosphate group as described herein; or may be absent.

R¹⁰⁰, R²⁰⁰, and R³⁰⁰ are each, independently, H (i.e., abasicnucleotides), adenine, guanine, cytosine and uracil, inosine, thymine,xanthine, hypoxanthine, nubularine, tubercidine, isoguanisine,2-aminoadenine, 6-methyl and other alkyl derivatives of adenine andguanine, 2-propyl and other alkyl derivatives of adenine and guanine,5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil,cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil,5-halouracil, 5-(2-aminopropyl)uracil, 5-amino allyl uracil, 8-halo,amino, thiol, thioalkyl, hydroxyl and other 8-substituted adenines andguanines, 5-trifluoromethyl and other 5-substituted uracils andcytosines, 7-methylguanine, 5-substituted pyrimidines, 6-azapyrimidinesand N-2, N-6 and O-6 substituted purines, including2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine,dihydrouracil, 3-deaza-5-azacytosine, 2-aminopurine, 5-alkyluracil,7-alkylguanine, 5-alkyl cytosine, 7-deazaadenine, 7-deazaguanine, N6,N6-dimethyladenine, 2,6-diaminopurine, 5-amino-allyl-uracil,N3-methyluracil substituted 1,2,4,-triazoles, 2-pyridinones,5-nitroindole, 3-nitropyrrole, 5-methoxyuracil, uracil-5-oxyacetic acid,5-methoxycarbonylmethyluracil, 5-methyl-2-thiouracil,5-methoxycarbonylmethyl-2-thiouracil, 5-methylaminomethyl-2-thiouracil,3-(3-amino-3carboxypropyl)uracil, 3-methylcytosine, 5-methylcytosine,N⁴-acetyl cytosine, 2-thiocytosine, N6-methyladenine,N6-isopentyladenine, 2-methylthio-N-6-isopentenyladenine,N-methylguanines, or O-alkylated bases.

x is 5-100, or chosen to comply with a length for an RNA agent describedherein; and g is 0-2.

Nuclease Resistant Monomers

The monomers and methods described herein can be used to prepare an RNA,e.g., an iRNA agent, that incorporates a nuclease resistant monomer(NRM), such as those described herein and those described in copending,co-owned U.S. Provisional Application Ser. No. 60/469,612, filed on May9, 2003, and International Application No. PCT/US04/07070, both of whichare hereby incorporated by reference.

An iRNA agent can include monomers which have been modified so as toinhibit degradation, e.g., by nucleases, e.g., endonucleases orexonucleases, found in the body of a subject. These monomers arereferred to herein as NRMs, or nuclease resistance promoting monomers ormodifications. In many cases these modifications will modulate otherproperties of the iRNA agent as well, e.g., the ability to interact witha protein, e.g., a transport protein, e.g., serum albumin, or a memberof the RISC(RNA-induced Silencing Complex), or the ability of the firstand second sequences to form a duplex with one another or to form aduplex with another sequence, e.g., a target molecule.

While not wishing to be bound by theory, it is believed thatmodifications of the sugar, base, and/or phosphate backbone in an iRNAagent can enhance endonuclease and exonuclease resistance, and canenhance interactions with transporter proteins and one or more of thefunctional components of the RISC complex. Preferred modifications arethose that increase exonuclease and endonuclease resistance and thusprolong the half-life of the iRNA agent prior to interaction with theRISC complex, but at the same time do not render the iRNA agentresistant to endonuclease activity in the RISC complex. Again, while notwishing to be bound by any theory, it is believed that placement of themodifications at or near the 3′ and/or 5′ end of antisense strands canresult in iRNA agents that meet the preferred nuclease resistancecriteria delineated above. Again, still while not wishing to be bound byany theory, it is believed that placement of the modifications at e.g.,the middle of a sense strand can result in iRNA agents that arerelatively less likely to undergo off-targeting.

Modifications described herein can be incorporated into anydouble-stranded RNA and RNA-like molecule described herein, e.g., aniRNA agent. An iRNA agent may include a duplex comprising a hybridizedsense and antisense strand, in which the antisense strand and/or thesense strand may include one or more of the modifications describedherein. The anti sense strand may include modifications at the 3′ endand/or the 5′ end and/or at one or more positions that occur 1-6 (e.g.,1-5, 1-4, 1-3, 1-2) nucleotides from either end of the strand. The sensestrand may include modifications at the 3′ end and/or the 5′ end and/orat any one of the intervening positions between the two ends of thestrand. The iRNA agent may also include a duplex comprising twohybridized antisense strands. The first and/or the second antisensestrand may include one or more of the modifications described herein.Thus, one and/or both antisense strands may include modifications at the3′ end and/or the 5′ end and/or at one or more positions that occur 1-6(e.g., 1-5, 1-4, 1-3, 1-2) nucleotides from either end of the strand.Particular configurations are discussed below.

Modifications that can be useful for producing iRNA agents that meet thepreferred nuclease resistance criteria delineated above can include oneor more of the following chemical and/or stereochemical modifications ofthe sugar, base, and/or phosphate backbone:

-   -   (i) chiral (S_(P)) thioates. Thus, preferred NRMs include        nucleotide dimers with an enriched or pure for a particular        chiral form of a modified phosphate group containing a        heteroatom at the nonbridging position, e.g., Sp or Rp, at the        position X, where this is the position normally occupied by the        oxygen. The atom at X can also be S, Se, Nr₂, or Br₃. When X is        S, enriched or chirally pure Sp linkage is preferred. Enriched        means at least 70, 80, 90, 95, or 99% of the preferred form.        Such NRMs are discussed in more detail below;

(ii) attachment of one or more cationic groups to the sugar, base,and/or the phosphorus atom of a phosphate or modified phosphate backbonemoiety. Thus, preferred NRMs include monomers at the terminal positionderivatized at a cationic group. As the 5′ end of an antisense sequenceshould have a terminal —OH or phosphate group this NRM is preferably notused at the 5′ end of an anti-sense sequence. The group should beattached at a position on the base which minimizes interference with Hbond formation and hybridization, e.g., away form the face whichinteracts with the complementary base on the other strand, e.g., at the5′ position of a pyrimidine or a 7-position of a purine. These arediscussed in more detail below;

(iii) nonphosphate linkages at the termini. Thus, preferred NRMs includeNon-phosphate linkages, e.g., a linkage of 4 atoms which confers greaterresistance to cleavage than does a phosphate bond. Examples include3′CH2—NCH₃—O—CH2-5′ and 3′CH2—NH—(O═)—CH2-5′;

(iv) 3′-bridging thiophosphates and 5′-bridging thiophosphates. Thus,preferred NRM's can included these structures;

(v) L-RNA, 2′-5′ linkages, inverted linkages, a-nucleosides. Thus, otherpreferred NRM's include: L nucleosides and dimeric nucleotides derivedfrom L-nucleosides; 2′-5′ phosphate, non-phosphate and modifiedphosphate linkages (e.g., thiophosphates, phosphoramidates andboronophosphates); dimers having inverted linkages, e.g., 3′-3′ or 5′-5′linkages; monomers having an alpha linkage at the 1′ site on the sugar,e.g., the structures described herein having an alpha linkage;

(vi) conjugate groups. Thus, preferred NRM's can include e.g., atargeting moiety or a conjugated ligand described herein conjugated withthe monomer, e.g., through the sugar, base, or backbone;

(vi) abasic linkages. Thus, preferred NRM's can include an abasicmonomer, e.g., an abasic monomer as described herein (e.g., anucleobaseless monomer); an aromatic or heterocyclic or polyheterocyclicaromatic monomer as described herein; and

(vii) 5′-phosphonates and 5′-phosphate prodrugs. Thus, preferred NRM'sinclude monomers, preferably at the terminal position, e.g., the 5′position, in which one or more atoms of the phosphate group isderivatized with a protecting group, which protecting group or groups,are removed as a result of the action of a component in the subject'sbody, e.g., a carboxyesterase or an enzyme present in the subject'sbody. E.g., a phosphate prodrug in which a carboxy esterase cleaves theprotected molecule resulting in the production of a thioate anion whichattacks a carbon adjacent to the O of a phosphate and resulting in theproduction of an unprotected phosphate.

One or more different NRM modifications can be introduced into an iRNAagent or into a sequence of an iRNA agent. An NRM modification can beused more than once in a sequence or in an iRNA agent. As some NRM'sinterfere with hybridization the total number incorporated, should besuch that acceptable levels of iRNA agent duplex formation aremaintained.

In some embodiments NRM modifications are introduced into the terminalthe cleavage site or in the cleavage region of a sequence (a sensestrand or sequence) which does not target a desired sequence or gene inthe subject. This can reduce off-target silencing.

Chiral S_(P) Thioates

A modification can include the alteration, e.g., replacement, of one orboth of the non-linking (X and Y) phosphate oxygens and/or of one ormore of the linking (W and Z) phosphate oxygens. Formula X below depictsa phosphate moiety linking two sugar/sugar surrogate-base moieties, SB₁and SB₂.

In certain embodiments, one of the non-linking phosphate oxygens in thephosphate backbone moiety (X and Y) can be replaced by any one of thefollowing: S, Se, BR₃ (R is hydrogen, alkyl, aryl, etc.), C (i.e., analkyl group, an aryl group, etc.), H, NR₂ (R is hydrogen, alkyl, aryl,etc.), or OR (R is alkyl or aryl). The phosphorus atom in an unmodifiedphosphate group is achiral. However, replacement of one of thenon-linking oxygens with one of the above atoms or groups of atomsrenders the phosphorus atom chiral; in other words a phosphorus atom ina phosphate group modified in this way is a stereogenic center. Thestereogenic phosphorus atom can possess either the “R” configuration(herein R_(P)) or the “S” configuration (herein S_(P)). Thus if 60% of apopulation of stereogenic phosphorus atoms have the R_(P) configuration,then the remaining 40% of the population of stereogenic phosphorus atomshave the S_(P) configuration.

In some embodiments, iRNA agents, having phosphate groups in which aphosphate non-linking oxygen has been replaced by another atom or groupof atoms, may contain a population of stereogenic phosphorus atoms inwhich at least about 50% of these atoms (e.g., at least about 60% ofthese atoms, at least about 70% of these atoms, at least about 80% ofthese atoms, at least about 90% of these atoms, at least about 95% ofthese atoms, at least about 98% of these atoms, at least about 99% ofthese atoms) have the S_(P) configuration. Alternatively, iRNA agentshaving phosphate groups in which a phosphate non-linking oxygen has beenreplaced by another atom or group of atoms may contain a population ofstereogenic phosphorus atoms in which at least about 50% of these atoms(e.g., at least about 60% of these atoms, at least about 70% of theseatoms, at least about 80% of these atoms, at least about 90% of theseatoms, at least about 95% of these atoms, at least about 98% of theseatoms, at least about 99% of these atoms) have the R_(P) configuration.In other embodiments, the population of stereogenic phosphorus atoms mayhave the S_(P) configuration and may be substantially free ofstereogenic phosphorus atoms having the R_(P) configuration. In stillother embodiments, the population of stereogenic phosphorus atoms mayhave the R_(P) configuration and may be substantially free ofstereogenic phosphorus atoms having the S_(P) configuration. As usedherein, the phrase “substantially free of stereogenic phosphorus atomshaving the R_(P) configuration” means that moieties containingstereogenic phosphorus atoms having the R_(P) configuration cannot bedetected by conventional methods known in the art (chiral HPLC, ¹H NMRanalysis using chiral shift reagents, etc.). As used herein, the phrase“substantially free of stereogenic phosphorus atoms having the S_(P)configuration” means that moieties containing stereogenic phosphorusatoms having the S_(P) configuration cannot be detected by conventionalmethods known in the art (chiral HPLC, ¹H NMR analysis using chiralshift reagents, etc.).

In a preferred embodiment, modified iRNA agents contain aphosphorothioate group, i.e., a phosphate groups in which a phosphatenon-linking oxygen has been replaced by a sulfur atom. In an especiallypreferred embodiment, the population of phosphorothioate stereogenicphosphorus atoms may have the S_(P) configuration and be substantiallyfree of stereogenic phosphorus atoms having the R_(P) configuration.

Phosphorothioates may be incorporated into iRNA agents using dimerse.g., formulas X-1 and X-2. The former can be used to introducephosphorothioate

at the 3′ end of a strand, while the latter can be used to introducethis modification at the 5′ end or at a position that occurs e.g., 1, 2,3, 4, 5, or 6 nucleotides from either end of the strand. In the aboveformulas, Y can be 2-cyanoethoxy, W and Z can be O, R_(2′) can be, e.g.,a substituent that can impart the C-3 endo configuration to the sugar(e.g., OH, F, OCH₃), DMT is dimethoxytrityl, and “BASE” can be anatural, unusual, or a universal base.

X-1 and X-2 can be prepared using chiral reagents or directing groupsthat can result in phosphorothioate-containing dimers having apopulation of stereogenic phosphorus atoms having essentially only theR_(P) configuration (i.e., being substantially free of the S_(P)configuration) or only the S_(P) configuration (i.e., beingsubstantially free of the R_(P) configuration). Alternatively, dimerscan be prepared having a population of stereogenic phosphorus atoms inwhich about 50% of the atoms have the R_(P) configuration and about 50%of the atoms have the S_(P) configuration. Dimers having stereogenicphosphorus atoms with the R_(P) configuration can be identified andseparated from dimers having stereogenic phosphorus atoms with the S_(P)configuration using e.g., enzymatic degradation and/or conventionalchromatography techniques.

Cationic Groups

Modifications can also include attachment of one or more cationic groupsto the sugar, base, and/or the phosphorus atom of a phosphate ormodified phosphate backbone moiety. A cationic group can be attached toany atom capable of substitution on a natural, unusual or universalbase. A preferred position is one that does not interfere withhybridization, i.e., does not interfere with the hydrogen bondinginteractions needed for base pairing. A cationic group can be attachede.g., through the C2′ position of a sugar or analogous position in acyclic or acyclic sugar surrogate. Cationic groups can include e.g.,protonated amino groups, derived from e.g., O-AMINE (AMINE=NH₂;alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino,heteroaryl amino, or diheteroaryl amino, ethylene diamine, polyamino);aminoalkoxy, e.g., O(CH₂)_(n)AMINE, (e.g., AMINE=NH₂; alkylamino,dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino,or diheteroaryl amino, ethylene diamine, polyamino); amino (e.g. NH₂;alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino,heteroaryl amino, diheteroaryl amino, or amino acid); orNH(CH₂CH₂NH)_(n)CH₂CH₂-AMINE (AMINE=NH₂; alkylamino, dialkylamino,heterocyclyl, arylamino, diaryl amino, heteroaryl amino, or diheteroarylamino).

Nonphosphate Linkages

Modifications can also include the incorporation of nonphosphatelinkages at the 5′ and/or 3′ end of a strand. Examples of nonphosphatelinkages which can replace the phosphate group include methylphosphonate, hydroxylamino, siloxane, carbonate, carboxymethyl,carbamate, amide, thioether, ethylene oxide linker, sulfonate,sulfonamide, thioformacetal, formacetal, oxime, methyleneimino,methylenemethylimino, methylenehydrazo, methylenedimethylhydrazo andmethyleneoxymethylimino. Preferred replacements include the methylphosphonate and hydroxylamino groups.

3′-Bridging Thiophosphates and 5′-Bridging Thiophosphates; Locked-RNA,2′-5′ Likages, Inverted Linkages, α-Nucleosides; Conjugate Groups;Abasic Linkages; and 5′-Phosphonates and 5′-Phosphate Prodrugs

Referring to formula X above, modifications can include replacement ofone of the bridging or linking phosphate oxygens in the phosphatebackbone moiety (W and Z). Unlike the situation where only one of X or Yis altered, the phosphorus center in the phosphorodithioates is achiralwhich precludes the formation of iRNA agents containing a stereogenicphosphorus atom.

Modifications can also include linking two sugars via a phosphate ormodified phosphate group through the 2′ position of a first sugar andthe 5′ position of a second sugar. Also contemplated are invertedlinkages in which both a first and second sugar are eached linkedthrough the respective 3′ positions. Modified RNA's can also include“abasic” sugars, which lack a nucleobase at C-1′. The sugar group canalso contain one or more carbons that possess the oppositestereochemical configuration than that of the corresponding carbon inribose. Thus, a modified iRNA agent can include nucleotides containinge.g., arabinose, as the sugar. In another subset of this modification,the natural, unusual, or universal base may have the α-configuration.Modifcations can also include L-RNA.

Modifications can also include 5′-phosphonates, e.g.,P(O)(O⁻)₂—X—C^(5′)-sugar (X=CH2, CF2, CHF and 5′-phosphate prodrugs,e.g., P(O)[OCH2CH2SC(O)R]₂CH₂C^(5′)-sugar. In the latter case, theprodrug groups may be decomposed via reaction first with carboxyesterases. The remaining ethyl thiolate group via intramolecular S_(N)2displacement can depart as episulfide to afford the underivatizedphosphate group.

Modification can also include the addition of conjugating groupsdescribed elsewhere herein, which are preferably attached to an iRNAagent through any amino group available for conjugation.

Nuclease resistant modifications include some which can be placed onlyat the terminus and others which can go at any position. Generally themodifications that can inhibit hybridization so it is preferably to usethem only in terminal regions, and preferable to not use them at thecleavage site or in the cleavage region of an sequence which targets asubject sequence or gene. The can be used anywhere in a sense sequence,provided that sufficient hybridization between the two sequences of theiRNA agent is maintained. In some embodiments it is desirable to put theNRM at the cleavage site or in the cleavage region of a sequence whichdoes not target a subject sequence or gene, as it can minimizeoff-target silencing.

In addition, an iRNA agent described herein can have an overhang whichdoes not form a duplex structure with the other sequence of the iRNAagent—it is an overhang, but it does hybridize, either with itself, orwith another nucleic acid, other than the other sequence of the iRNAagent.

In most cases, the nuclease-resistance promoting modifications will bedistributed differently depending on whether the sequence will target asequence in the subject (often referred to as an antisense sequence) orwill not target a sequence in the subject (often referred to as a sensesequence). If a sequence is to target a sequence in the subject,modifications which interfer with or inhibit endonuclease cleavageshould not be inserted in the region which is subject to RISC mediatedcleavage, e.g., the cleavage site or the cleavage region (As describedin Elbashir et al., 2001, Genes and Dev. 15: 188, hereby incorporated byreference, cleavage of the target occurs about in the middle of a 20 or21 nt guide RNA, or about 10 or 11 nucleotides upstream of the firstnucleotide which is complementary to the guide sequence. As used hereincleavage site refers to the nucleotide on either side of the cleavagesite, on the target or on the iRNA agent strand which hybridizes to it.Cleavage region means an nucleotide with 1, 2, or 3 nucletides of thecleave site, in either direction.)

Such modifications can be introduced into the terminal regions, e.g., atthe terminal position or with 2, 3, 4, or 5 positions of the terminus,of a sequence which targets or a sequence which does not target asequence in the subject.

An iRNA agent can have a first and a second strand chosen from thefollowing:

a first strand which does not target a sequence and which has an NRMmodification at or within 1, 2, 3, 4, 5, or 6 positions from the 3′ end;

a first strand which does not target a sequence and which has an NRMmodification at or within 1, 2, 3, 4, 5, or 6 positions from the 5′ end;

a first strand which does not target a sequence and which has an NRMmodification at or within 1, 2, 3, 4, 5, or 6 positions from the 3′ endand which has a NRM modification at or within 1, 2, 3, 4, 5, or 6positions from the 5′ end;

a first strand which does not target a sequence and which has an NRMmodification at the cleavage site or in the cleavage region;

a first strand which does not target a sequence and which has an NRMmodification at the cleavage site or in the cleavage region and one ormore of an NRM modification at or within 1, 2, 3, 4, 5, or 6 positionsfrom the 3′ end, a NRM modification at or within 1, 2, 3, 4, 5, or 6positions from the 5′ end, or NRM modifications at or within 1, 2, 3, 4,5, or 6 positions from both the 3′ and the 5′ end; and

a second strand which targets a sequence and which has an NRMmodification at or within 1, 2, 3, 4, 5, or 6 positions from the 3′ end;

a second strand which targets a sequence and which has an NRMmodification at or within 1, 2, 3, 4, 5, or 6 positions from the 5′ end(5′ end NRM modifications are preferentially not at the terminus butrather at a position 1, 2, 3, 4, 5, or 6 away from the 5′ terminus of anantisense strand);

a second strand which targets a sequence and which has an NRMmodification at or within 1, 2, 3, 4, 5, or 6 positions from the 3′ endand which has a NRM modification at or within 1, 2, 3, 4, 5 or 6positions from the 5′ end;

a second strand which targets a sequence and which preferably does nothave an an NRM modification at the cleavage site or in the cleavageregion;

a second strand which targets a sequence and which does not have an NRMmodification at the cleavage site or in the cleavage region and one ormore of an NRM modification at or within 1, 2, 3, 4, 5, or 6 positionsfrom the 3′ end, a NRM modification at or within 1, 2, 3, 4, 5, or 6positions from the 5′ end, or NRM modifications at or within 1, 2, 3, 4,5, or 6 positions from both the 3′ and the 5′ end (5′ end NRMmodifications are preferentially not at the terminus but rather at aposition 1, 2, 3, 4, 5, or 6 away from the 5′ terminus of an antisensestrand).

An iRNA agent can also target two sequences and can have a first andsecond strand chosen from:

a first strand which targets a sequence and which has an NRMmodification at or within 1, 2, 3, 4, 5, or 6 positions from the 3′ end;

a first strand which targets a sequence and which has an NRMmodification at or within 1, 2, 3, 4, 5, or 6 positions from the 5′ end(5′ end NRM modifications are preferentially not at the terminus butrather at a position 1, 2, 3, 4, 5, or 6 away from the 5′ terminus of anantisense strand);

a first strand which targets a sequence and which has an NRMmodification at or within 1, 2, 3, 4, 5, or 6 positions from the 3′ endand which has a NRM modification at or within 1, 2, 3, 4, 5, or 6positions from the 5′ end;

a first strand which targets a sequence and which preferably does nothave an an NRM modification at the cleavage site or in the cleavageregion;

a first strand which targets a sequence and which dose not have an NRMmodification at the cleavage site or in the cleavage region and one ormore of an NRM modification at or within 1, 2, 3, 4, 5, or 6 positionsfrom the 3′ end, a NRM modification at or within 1, 2, 3, 4, 5, or 6positions from the 5′ end, or NRM modifications at or within 1, 2, 3, 4,5, or 6 positions from both the 3′ and the 5′ end (5′ end NRMmodifications are preferentially not at the terminus but rather at aposition 1, 2, 3, 4, 5, or 6 away from the 5′ terminus of an antisensestrand) and

a second strand which targets a sequence and which has an NRMmodification at or within 1, 2, 3, 4, 5, or 6 positions from the 3′ end;

a second strand which targets a sequence and which has an NRMmodification at or within 1, 2, 3, 4, 5, or 6 positions from the 5′ end(5′ end NRM modifications are preferentially not at the terminus butrather at a position 1, 2, 3, 4, 5, or 6 away from the 5′ terminus of anantisense strand);

a second strand which targets a sequence and which has an NRMmodification at or within 1, 2, 3, 4, 5, or 6 positions from the 3′ endand which has a NRM modification at or within 1, 2, 3, 4, 5, or 6positions from the 5′ end;

a second strand which targets a sequence and which preferably does nothave an an NRM modification at the cleavage site or in the cleavageregion;

a second strand which targets a sequence and which dose not have an NRMmodification at the cleavage site or in the cleavage region and one ormore of an NRM modification at or within 1, 2, 3, 4, 5, or 6 positionsfrom the 3′ end, a NRM modification at or within 1, 2, 3, 4, 5, or 6positions from the 5′ end, or NRM modifications at or within 1, 2, 3, 4,5, or 6 positions from both the 3′ and the 5′ end (5′ end NRMmodifications are preferentially not at the terminus but rather at aposition 1, 2, 3, 4, 5, or 6 away from the 5′ terminus of an antisensestrand).

Ribose Mimics

The monomers and methods described herein can be used to prepare an RNA,e.g., an iRNA agent, that incorporates a ribose mimic, such as thosedescribed herein and those described in copending co-owned U.S.Provisional Application Ser. No. 60/454,962, filed on Mar. 13, 2003, andInternational Application No. PCT/US04/07070, both of which are herebyincorporated by reference.

Thus, an aspect of the invention features an iRNA agent that includes asecondary hydroxyl group, which can increase efficacy and/or confernuclease resistance to the agent. Nucleases, e.g., cellular nucleases,can hydrolyze nucleic acid phosphodiester bonds, resulting in partial orcomplete degradation of the nucleic acid. The secondary hydroxy groupconfers nuclease resistance to an iRNA agent by rendering the iRNA agentless prone to nuclease degradation relative to an iRNA which lacks themodification. While not wishing to be bound by theory, it is believedthat the presence of a secondary hydroxyl group on the iRNA agent canact as a structural mimic of a 3′ ribose hydroxyl group, thereby causingit to be less susceptible to degradation.

The secondary hydroxyl group refers to an “OH” radical that is attachedto a carbon atom substituted by two other carbons and a hydrogen. Thesecondary hydroxyl group that confers nuclease resistance as describedabove can be part of any acyclic carbon-containing group. The hydroxylmay also be part of any cyclic carbon-containing group, and preferablyone or more of the following conditions is met (1) there is no ribosemoiety between the hydroxyl group and the terminal phosphate group or(2) the hydroxyl group is not on a sugar moiety which is coupled to abase. The hydroxyl group is located at least two bonds (e.g., at leastthree bonds away, at least four bonds away, at least five bonds away, atleast six bonds away, at least seven bonds away, at least eight bondsaway, at least nine bonds away, at least ten bonds away, etc.) from theterminal phosphate group phosphorus of the iRNA agent. In preferredembodiments, there are five intervening bonds between the terminalphosphate group phosphorus and the secondary hydroxyl group.

Preferred iRNA agent delivery modules with five intervening bondsbetween the terminal phosphate group phosphorus and the secondaryhydroxyl group have the following structure (see formula Y below):

Referring to formula Y, A is an iRNA agent, including any iRNA agentdescribed herein. The iRNA agent may be connected directly or indirectly(e.g., through a spacer or linker) to “W” of the phosphate group. Thesespacers or linkers can include e.g., —(CH₂)_(n)—, —(CH₂)_(n)N—,—(CH₂)_(n)O—, —(CH₂)_(n)S—, O(CH₂CH₂O)_(n)CH₂CH₂OH (e.g., n=3 or 6),abasic sugars, amide, carboxy, amine, oxyamine, oxyimine, thioether,disulfide, thiourea, sulfonamide, or morpholino, or biotin andfluorescein reagents.

The iRNA agents can have a terminal phosphate group that is unmodified(e.g., W, X, Y, and Z are O) or modified. In a modified phosphate group,W and Z can be independently NH, O, or S; and X and Y can beindependently S, Se, BH₃ ⁻, C₁-C₆ alkyl, C₆-C₁₀ aryl, H, O, O⁻, alkoxyor amino (including alkylamino, arylamino, etc.). Preferably, W, X and Zare O and Y is S.

R₁ and R₃ are each, independently, hydrogen; or C₁-C₁₀₀ alkyl,optionally substituted with hydroxyl, amino, halo, phosphate or sulfateand/or may be optionally inserted with N, O, S, alkenyl or alkynyl.

R₂ is hydrogen; C₁-C₁₀₀ alkyl, optionally substituted with hydroxyl,amino, halo, phosphate or sulfate and/or may be optionally inserted withN, O, S, alkenyl or alkynyl; or, when n is 1, R₂ may be taken togetherwith with R₄ or R₆ to form a ring of 5-12 atoms.

R₄ is hydrogen; C₁-C₁₀₀ alkyl, optionally substituted with hydroxyl,amino, halo, phosphate or sulfate and/or may be optionally inserted withN, O, S, alkenyl or alkynyl; or, when n is 1, R₄ may be taken togetherwith with R₂ or R₅ to form a ring of 5-12 atoms.

R₅ is hydrogen, C₁-C₁₀₀ alkyl optionally substituted with hydroxyl,amino, halo, phosphate or sulfate and/or may be optionally inserted withN, O, S, alkenyl or alkynyl; or, when n is 1, R₅ may be taken togetherwith with R₄ to form a ring of 5-12 atoms.

R₆ is hydrogen, C₁-C₁₀₀ alkyl, optionally substituted with hydroxyl,amino, halo, phosphate or sulfate and/or may be optionally inserted withN, O, S, alkenyl or alkynyl, or, when n is 1, R₆ may be taken togetherwith with R₂ to form a ring of 6-10 atoms;

R₇ is hydrogen, C₁-C₁₀₀ alkyl, or C(O)(CH₂)_(q)C(O)NHR₉; T is hydrogenor a functional group; n and q are each independently 1-100; R₈ isC₁-C₁₀ alkyl or C₆-C₁₀ aryl; and R₉ is hydrogen, C1-C10 alkyl, C6-C10aryl or a solid support agent.

Preferred embodiments may include one of more of the following subsetsof iRNA agent delivery modules.

In one subset of RNAi agent delivery modules, Δ can be connecteddirectly or indirectly through a terminal 3′ or 5′ ribose sugar carbonof the RNA agent.

In another subset of RNAi agent delivery modules, X, W, and Z are O andY is S.

In still yet another subset of RNAi agent delivery modules, n is 1, andR₂ and R₆ are taken together to form a ring containing six atoms and R₄and R₅ are taken together to form a ring containing six atoms.Preferably, the ring system is a trans-decalin. For example, the RNAiagent delivery module of this subset can include a compound of Formula(Y-1):

The functional group can be, for example, a targeting group (e.g., asteroid or a carbohydrate), a reporter group (e.g., a fluorophore), or alabel (an isotopically labelled moiety). The targeting group can furtherinclude protein binding agents, endothelial cell targeting groups (e.g.,RGD peptides and mimetics), cancer cell targeting groups (e.g., folateVitamin B12, Biotin), bone cell targeting groups (e.g., bisphosphonates,polyglutamates, polyaspartates), multivalent mannose (for e.g.,macrophage testing), lactose, galactose, N-acetyl-galactosamine,monoclonal antibodies, glycoproteins, lectins, melanotropin, orthyrotropin.

As can be appreciated by the skilled artisan, methods of synthesizingthe compounds of the formulae herein will be evident to those ofordinary skill in the art. The synthesized compounds can be separatedfrom a reaction mixture and further purified by a method such as columnchromatography, high pressure liquid chromatography, orrecrystallization. Additionally, the various synthetic steps may beperformed in an alternate sequence or order to give the desiredcompounds. Synthetic chemistry transformations and protecting groupmethodologies (protection and deprotection) useful in synthesizing thecompounds described herein are known in the art and include, forexample, those such as described in R. Larock, Comprehensive OrganicTransformations, VCH Publishers (1989); T. W. Greene and P. G. M. Wuts,Protective Groups in Organic Synthesis, 2d. Ed., John Wiley and Sons(1991); L. Fieser and M. Fieser, Fieser and Fieser's Reagents forOrganic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed.,Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons(1995), and subsequent editions thereof.

Physiological Effects

The iRNA agents described herein can be designed such that determiningtherapeutic toxicity is made easier by the complementarity of the iRNAagent with both a human and a non-human animal sequence. By thesemethods, an iRNA agent can consist of a sequence that is fullycomplementary to a nucleic acid sequence from a human and a nucleic acidsequence from at least one non-human animal, e.g., a non-human mammal,such as a rodent, ruminant or primate. For example, the non-human mammalcan be a mouse, rat, dog, pig, goat, sheep, cow, monkey, Pan paniscus,Pan troglodytes, Macaca mulatto, or Cynomolgus monkey. The sequence ofthe iRNA agent could be complementary to sequences within homologousgenes, e.g., oncogenes or tumor suppressor genes, of the non-humanmammal and the human. By determining the toxicity of the iRNA agent inthe non-human mammal, one can extrapolate the toxicity of the iRNA agentin a human. For a more strenuous toxicity test, the iRNA agent can becomplementary to a human and more than one, e.g., two or three or more,non-human animals.

The methods described herein can be used to correlate any physiologicaleffect of an iRNA agent on a human, e.g., any unwanted effect, such as atoxic effect, or any positive, or desired effect.

iRNA Conjugates

An iRNA agent can be coupled, e.g., covalently coupled, to a secondagent. For example, an iRNA agent used to treat a particular disordercan be coupled to a second therapeutic agent, e.g., an agent other thanthe iRNA agent. The second therapeutic agent can be one which isdirected to the treatment of the same disorder. For example, in the caseof an iRNA used to treat a disorder characterized by unwanted cellproliferation, e.g., cancer, the iRNA agent can be coupled to a secondagent which has an anti-cancer effect. For example, it can be coupled toan agent which stimulates the immune system, e.g., a CpG motif, or moregenerally an agent that activates a toll-like receptor and/or increasesthe production of gamma interferon.

iRNA Production

An iRNA can be produced, e.g., in bulk, by a variety of methods.Exemplary methods include: organic synthesis and RNA cleavage, e.g., invitro cleavage.

Organic Synthesis. An iRNA can be made by separately synthesizing eachrespective strand of a double-stranded RNA molecule. The componentstrands can then be annealed.

A large bioreactor, e.g., the OligoPilot II from Pharmacia Biotec AB(Uppsala Sweden), can be used to produce a large amount of a particularRNA strand for a given iRNA. The OligoPilotII reactor can efficientlycouple a nucleotide using only a 1.5 molar excess of a phosphoramiditenucleotide. To make an RNA strand, ribonucleotides amidites are used.Standard cycles of monomer addition can be used to synthesize theoligonucleotide strands for the iRNA. Typically, the two complementarystrands are produced separately and then annealed, e.g., after releasefrom the solid support and deprotection.

Organic synthesis can be used to produce a discrete iRNA species. Thecomplementarity of the species to the target gene can be preciselyspecified. For example, the species may be complementary to a regionthat includes a polymorphism, e.g., a single nucleotide polymorphism.Further the location of the polymorphism can be precisely defined. Insome embodiments, the polymorphism is located in an internal region,e.g., at least 4, 5, 7, or 9 nucleotides from one or both of thetermini.

dsRNA Cleavage. iRNAs can also be made by cleaving a larger ds iRNA. Thecleavage can be mediated in vitro or in vivo. For example, to produceiRNAs by cleavage in vitro, the following method can be used:

In vitro transcription. dsRNA is produced by transcribing a nucleic acid(DNA) segment in both directions. For example, the HiScribe™ RNAitranscription kit (New England Biolabs) provides a vector and a methodfor producing a dsRNA for a nucleic acid segment that is cloned into thevector at a position flanked on either side by a T7 promoter. Separatetemplates are generated for T7 transcription of the two complementarystrands for the dsRNA. The templates are transcribed in vitro byaddition of T7 RNA polymerase and dsRNA is produced. Similar methodsusing PCR and/or other RNA polymerases (e.g., T3 or SP6 polymerase) canalso be used. In one embodiment, RNA generated by this method iscarefully purified to remove endotoxins that may contaminatepreparations of the recombinant enzymes.

In vitro cleavage. dsRNA is cleaved in vitro into iRNAs, for example,using a Dicer or comparable RNAse III-based activity. For example, thedsRNA can be incubated in an in vitro extract from Drosophila or usingpurified components, e.g. a purified RNAse or RISC complex. See, e.g.,Ketting et al. Genes Dev 2001 Oct. 15; 15(20):2654-9. and HammondScience 2001 Aug. 10; 293(5532):1146-50.

dsRNA cleavage generally produces a plurality of iRNA species, eachbeing a particular 21 to 23 nt fragment of a source dsRNA molecule. Forexample, iRNAs that include sequences complementary to overlappingregions and adjacent regions of a source dsRNA molecule may be present.

Regardless of the method of synthesis, the iRNA preparation can beprepared in a solution (e.g., an aqueous and/or organic solution) thatis appropriate for formulation. For example, the iRNA preparation can beprecipitated and redissolved in pure double-distilled water, andlyophilized. The dried iRNA can then be resuspended in a solutionappropriate for the intended formulation process.

Synthesis of modified and nucleotide surrogate iRNA agents is discussedbelow.

Formulation

The iRNA agents described herein can be formulated for administration toa subject.

For ease of exposition, the formulations, compositions, and methods inthis section are discussed largely with regard to unmodified iRNAagents. It should be understood, however, that these formulations,compositions, and methods can be practiced with other iRNA agents, e.g.,modified iRNA agents, and such practice is within the invention.

A formulated iRNA composition can assume a variety of states. In someexamples, the composition is at least partially crystalline, uniformlycrystalline, and/or anhydrous (e.g., less than 80, 50, 30, 20, or 10%water). In another example, the iRNA is in an aqueous phase, e.g., in asolution that includes water.

The aqueous phase or the crystalline compositions can, e.g., beincorporated into a delivery vehicle, e.g., a liposome (particularly forthe aqueous phase) or a particle (e.g., a microparticle as can beappropriate for a crystalline composition). Generally, the iRNAcomposition is formulated in a manner that is compatible with theintended method of administration.

In particular embodiments, the composition is prepared by at least oneof the following methods: spray drying, lyophilization, vacuum drying,evaporation, fluid bed drying, or a combination of these techniques; orsonication with a lipid, freeze-drying, condensation and otherself-assembly.

An iRNA preparation can be formulated in combination with another agent,e.g., another therapeutic agent or an agent that stabilizes a iRNA,e.g., a protein that complexes with iRNA to form an iRNP. Still otheragents include chelators, e.g., EDTA (e.g., to remove divalent cationssuch as Mg²⁺), salts, RNAse inhibitors (e.g., a broad specificity RNAseinhibitor such as RNAsin) and so forth.

In one embodiment, the iRNA preparation includes another iRNA agent,e.g., a second iRNA agent that can mediate RNAi with respect to a secondgene, or with respect to the same gene. Still other preparations caninclude at least three, five, ten, twenty, fifty, or a hundred or moredifferent iRNA species. Such iRNAs can mediated RNAi with respect to asimilar number of different genes.

In one embodiment, the iRNA preparation includes at least a secondtherapeutic agent (e.g., an agent other than an RNA or a DNA).

In some embodiments, an iRNA agent, e.g., a double-stranded iRNA agent,or siRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which canbe processed into an siRNA agent, or a DNA which encodes an iRNA agent,e.g., a double-stranded iRNA agent, or siRNA agent, or precursorthereof) is formulated to target a particular cell. For example, aliposome or particle or other structure that includes a iRNA can alsoinclude a targeting moiety that recognizes a specific molecule on atarget cell. The targeting moiety can be a molecule with a specificaffinity for a target cell. Targeting moieties can include antibodiesdirected against a protein found on the surface of a target cell, or theligand or a receptor-binding portion of a ligand for a molecule found onthe surface of a target cell.

In one embodiment, the targeting moiety is attached to a liposome. Forexample, U.S. Pat. No. 6,245,427 describes a method for targeting aliposome using a protein or peptide. In another example, a cationiclipid component of the liposome is derivatized with a targeting moiety.For example, WO 96/37194 describes convertingN-glutaryldioleoylphosphatidyl ethanolamine to a N-hydroxysuccinimideactivated ester. The product was then coupled to an RGD peptide.Additional targeting methods are described elsewhere herein.

Pharmaceutical Compositions

In one embodiment, the invention relates to a pharmaceutical compositioncontaining a modified iRNA agent, as described in the precedingsections, and a pharmaceutically acceptable carrier, as described below.A pharmaceutical composition including the modified iRNA agent is usefulfor treating a disease caused by expression of a target gene. In thisaspect of the invention, the iRNA agent of the invention is formulatedas described below. The pharmaceutical composition is administered in adosage sufficient to inhibit expression of the target gene.

The pharmaceutical compositions of the present invention areadministered in dosages sufficient to inhibit the expression or activityof the target gene. Exemplary dosages and routes of administration aredescribed below.

The pharmaceutical compositions can include encapsulated formulations toprotect the iRNA agent against rapid elimination from the body, such asa controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811; PCT publication WO 91/06309; and European patent publicationEP-A-43075, which are incorporated by reference herein.

Toxicity and therapeutic efficacy of iRNA agent can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD50/ED50.iRNA agents that exhibit high therapeutic indices are preferred.

Advances in mouse genetics have generated a number of mouse models forthe study of various human diseases. For example, mouse repositories canbe found at The Jackson Laboratory, Charles River Laboratories, Taconic,Harlan, Mutant Mouse Regional Resource Centers (MMRRC) National Networkand at the European Mouse Mutant Archive. Such models may be used for invivo testing of iRNA agent, as well as for determining a therapeuticallyeffective dose.

The data obtained from cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosages ofcompositions of the invention are preferably within a range ofcirculating concentrations that include the ED50 with little or notoxicity. The dosage may vary within this range depending upon thedosage form employed and the route of administration utilized. For anyiRNA agent used in the method of the invention, the therapeuticallyeffective dose can be estimated initially from cell culture assays. Adose may be formulated in animal models to achieve a circulating plasmaconcentration range of the iRNA agent or, when appropriate, of thepolypeptide product of a target sequence (e.g., achieving a decreasedconcentration of the polypeptide) that includes the IC50 (i.e., theconcentration of the test iRNA agent which achieves a half-maximalinhibition of symptoms) as determined in cell culture. Such informationcan be used to more accurately determine useful doses in humans. Levelsin plasma may be measured, for example, by high performance liquidchromatography.

In addition to their administration individually or as a plurality, iRNAagents relating to the invention can be administered in combination withother known agents effective in treating viral infections and diseases.In any event, the administering physician can adjust the amount andtiming of iRNA agent administration on the basis of results observedusing standard measures of efficacy known in the art or describedherein.

Methods for Treating Diseases Caused by Expression of a Target Gene

In one embodiment, the invention relates to a method for treating asubject having a disease or at risk of developing a disease caused bythe expression of a target gene. In this embodiment, iRNA agents can actas novel therapeutic agents for controlling one or more of cellularproliferative and/or differentiative disorders, disorders associatedwith bone metabolism, immune disorders, hematopoietic disorders,cardiovascular disorders, liver disorders, viral diseases, or metabolicdisorders. The method includes administering a pharmaceuticalcomposition of the invention to the patient (e.g., a human), such thatexpression of the target gene is silenced. Because of their highefficiency and specificity, the iRNA agent of the present inventionspecifically target mRNA of target genes of diseased cells and tissues,as described below, and at surprisingly low dosages. The pharmaceuticalcompositions are formulated as described in the preceding section, whichis hereby incorporated by reference herein.

Examples of genes which can be targeted for treatment include, withoutlimitation, an oncogene (Hanahan, D. and R. A. Weinberg, Cell (2000)100:57; and Yokota, J., Carcinogenesis (2000) 21(3):497-503); a cytokinegene (Rubinstein, M., et al., Cytokine Growth Factor Rev. (1998)9(2):175-81); a idiotype (Id) protein gene (Benezra, R., et al.,Oncogene (2001) 20(58):8334-41; Norton, J. D., J. Cell Sci. (2000)113(22):3897-905); a prion gene (Prusiner, S. B., et al., Cell (1998)93(3):337-48; Safar, J., and S. B. Prusiner, Prog. Brain Res. (1998)117:421-34); a gene that expresses molecules that induce angiogenesis(Gould, V. E. and B. M. Wagner, Hum. Pathol. (2002) 33(11):1061-3);adhesion molecules (Chothia, C. and E. Y. Jones, Annu. Rev. Biochem.(1997) 66:823-62; Parise, L. V., et al., Semin. Cancer Biol. (2000)10(6):407-14); cell surface receptors (Deller, M. C., and Y. E. Jones,Curr. Opin. Struct. Biol. (2000) 10(2):213-9); genes of proteins thatare involved in metastasizing and/or invasive processes (Boyd, D.,Cancer Metastasis Rev. (1996) 15(1):77-89; Yokota, J., Carcinogenesis(2000) 21(3):497-503); genes of proteases as well as of molecules thatregulate apoptosis and the cell cycle (Matrisian, L. M., Curr. Biol.(1999) 9(20):R776-8; Krepela, E., Neoplasma (2001) 48(5):332-49; Basbaumand Werb, Curr. Opin. Cell Biol. (1996) 8:731-738; Birkedal-Hansen, etal., Crit. Rev. Oral Biol. Med. (1993) 4:197-250; Mignatti and Rifkin,Physiol. Rev. (1993) 73:161-195; Stetler-Stevenson, et al., Annu. Rev.Cell Biol. (1993) 9:541-573; Brinkerhoff, E., and L. M. Matrisan, NatureReviews (2002) 3:207-214; Strasser, A., et al., Annu. Rev. Biochem.(2000) 69:217-45; Chao, D. T. and S. J. Korsmeyer, Annu. Rev. Immunol.(1998) 16:395-419; Mullauer, L., et al., Mutat. Res. (2001)488(3):211-31; Fotedar, R., et al., Prog. Cell Cycle Res. (1996)2:147-63; Reed, J. C., Am. J. Pathol. (2000) 157(5):1415-30; D'Ari, R.,Bioassays (2001) 23(7):563-5); genes that express the EGF receptor;Mendelsohn, J. and J. Baselga, Oncogene (2000) 19(56):6550-65; Normanno,N., et al., Front. Biosci. (2001) 6:D685-707); and the multi-drugresistance 1 gene, MDR1 gene (Childs, S., and V. Ling, Imp. Adv. Oncol.(1994) 21-36).

In the prevention of disease, the target gene may be one which isrequired for initiation or maintenance of the disease, or which has beenidentified as being associated with a higher risk of contracting thedisease. In the treatment of disease, the iRNA agent can be brought intocontact with the cells or tissue exhibiting the disease. For example,iRNA agent substantially identical to all or part of a mutated geneassociated with cancer, or one expressed at high levels in tumor cells,may be brought into contact with or introduced into a cancerous cell ortumor gene.

Examples of cellular proliferative and/or differentiative disordersinclude cancer, e.g., a carcinoma, sarcoma, metastatic disorder orhematopoietic neoplastic disorder, such as a leukemia. A metastatictumor can arise from a multitude of primary tumor types, including butnot limited to those of prostate, colon, lung, breast and liver origin.As used herein, the terms “cancer,” “hyperproliferative,” and“neoplastic” refer to cells having the capacity for autonomous growth,i.e., an abnormal state or condition characterized by rapidlyproliferating cell growth. These terms are meant to include all types ofcancerous growths or oncogenic processes, metastatic tissues ormalignantly transformed cells, tissues, or organs, irrespective ofhistopathologic type or stage of invasiveness. Proliferative disordersalso include hematopoietic neoplastic disorders, including diseasesinvolving hyperplastic/neoplastic cells of hematopoietic origin, e.g.,arising from myeloid, lymphoid or erythroid lineages, or precursor cellsthereof.

The pharmaceutical compositions of the present invention can also beused to treat a variety of immune disorders, in particular thoseassociated with overexpression or aberrant expression of a gene orexpression of a mutant gene. Examples of hematopoietic disorders ordiseases include, without limitation, autoimmune diseases (including,for example, diabetes mellitus, arthritis (including rheumatoidarthritis, juvenile rheumatoid arthritis, osteoarthritis, psoriaticarthritis), multiple sclerosis, encephalomyelitis, myasthenia gravis,systemic lupus erythematosis, automimmune thyroiditis, dermatitis(including atopic dermatitis and eczematous dermatitis), psoriasis,Sjogren's Syndrome, Crohn's disease, aphthous ulcer, iritis,conjunctivitis, keratoconjunctivitis, ulcerative colitis, asthma,allergic asthma, cutaneous lupus erythematosus, scleroderma, vaginitis,proctitis, drug eruptions, leprosy reversal reactions, erythema nodosumleprosum, autoimmune uveitis, allergic encephalomyelitis, acutenecrotizing hemorrhagic encephalopathy, idiopathic bilateral progressivesensorineural hearing, loss, aplastic anemia, pure red cell anemia,idiopathic thrombocytopenia, polychondritis, Wegener's granulomatosis,chronic active hepatitis, Stevens-Johnson syndrome, idiopathic sprue,lichen planus, Graves' disease, sarcoidosis, primary biliary cirrhosis,uveitis posterior, and interstitial lung fibrosis), graft-versus-hostdisease, cases of transplantation, and allergy.

In another embodiment, the invention relates to methods for treatingviral diseases, including but not limited to hepatitis C, hepatitis B,herpes simplex virus (HSV), HIV-AIDS, poliovirus, and smallpox virus.iRNA agent of the invention are prepared as described herein to targetexpressed sequences of a virus, thus ameliorating viral activity andreplication. The iRNA agents can be used in the treatment and/ordiagnosis of viral infected tissue, both animal and plant. Also, suchiRNA agent can be used in the treatment of virus-associated carcinoma,such as hepatocellular cancer.

For example, the iRNA agent of the present invention are useful fortreating a subject having an infection or a disease associated with thereplication or activity of a (+) strand RNA virus having a 3′-UTR, suchas HCV. In this embodiment, the iRNA agent can act as novel therapeuticagents for inhibiting replication of the virus. The method includesadministering a pharmaceutical composition of the invention to thepatient (e.g., a human), such that viral replication is inhibited.Examples of (+) strand RNA viruses which can be targeted for inhibitioninclude, without limitation, picomaviruses, caliciviruses, nodaviruses,coronaviruses, arteriviruses, flaviviruses, and togaviruses. Examples ofpicomaviruses include enterovirus (poliovirus 1), rhinovirus (humanrhinovirus 1A), hepatovirus (hepatitis A virus), cardiovirus(encephalomyocarditis virus), aphthovirus (foot-and-mouth disease virusO), and parechovirus (human echovirus 22). Examples of calicivirusesinclude vesiculovirus (swine vesicular exanthema virus), lagovirus(rabbit hemorrhagic disease virus), “Norwalk-like viruses” (Norwalkvirus), “Sapporo-like viruses” (Sapporo virus), and “hepatitis E-likeviruses” (hepatitis E virus). Betanodavirus (striped jack nervousnecrosis virus) is the representative nodavirus. Coronaviruses includecoronavirus (avian infections bronchitis virus) and torovirus (Bernevirus). Arterivirus (equine arteritis virus) is the representativearteriviridus. Togavirises include alphavirus (Sindbis virus) andrubivirus (Rubella virus). Finally, the flaviviruses include flavivirus(Yellow fever virus), pestivirus (bovine diarrhea virus), andhepacivirus (hepatitis C virus). In a preferred embodiment, the virus ishepacivirus, the hepatitis C virus. Although the foregoing listexemplifies vertebrate viruses, the present invention encompasses thecompositions and methods for treating infections and diseases caused byany (+) strand RNA virus having a 3′-UTR, regardless of the host. Forexample, the invention encompasses the treatment of plant diseasescaused by sequiviruses, comoviruses, potyviruses, sobemovirus,luteoviruses, tombusviruses, tobavirus, tobravirus, bromoviruses, andclosteroviruses.

Treatment Methods and Routes of Delivery

A composition that includes an iRNA agent can be delivered to a subjectby a variety of routes. Exemplary routes include intrathecal,parenchymal, intravenous, nasal, oral, and ocular delivery.

An iRNA agent can be incorporated into pharmaceutical compositionssuitable for administration. For example, compositions can include oneor more species of an iRNA agent and a pharmaceutically acceptablecarrier. As used herein the language “pharmaceutically acceptablecarrier” is intended to include any and all solvents, dispersion media,coatings, antibacterial and antifungal agents, isotonic and absorptiondelaying agents, and the like, compatible with pharmaceuticaladministration. The use of such media and agents for pharmaceuticallyactive substances is well known in the art. Except insofar as anyconventional media or agent is incompatible with the active compound,use thereof in the compositions is contemplated. Supplementary activecompounds can also be incorporated into the compositions.

The pharmaceutical compositions featured in the present invention may beadministered in a number of ways depending upon whether local orsystemic treatment is desired and upon the area to be treated.Administration may be topical (including ophthalmic, intranasal,transdermal), oral or parenteral. Parenteral administration includesintravenous drip, subcutaneous, intraperitoneal or intramuscularinjection, or intrathecal or intraventricular administration.

The route of delivery can be dependent on the disorder of the patient.In general, the delivery of the iRNA agents can achieve systemicdelivery into the subject.

Formulations for parenteral administration may include sterile aqueoussolutions which may also contain buffers, diluents and other suitableadditives. For intravenous use, the total concentration of solutesshould be controlled to render the preparation isotonic.

Administration can be provided by the subject or by another person,e.g., a another caregiver. A caregiver can be any entity involved withproviding care to the human: for example, a hospital, hospice, doctor'soffice, outpatient clinic; a healthcare worker such as a doctor, nurse,or other practitioner; or a spouse or guardian, such as a parent. Themedication can be provided in measured doses or in a dispenser whichdelivers a metered dose.

The subject can also be monitored for an improvement or stabilization ofdisease symptoms.

The term “therapeutically effective amount” is the amount present in thecomposition that is needed to provide the desired level of drug in thesubject to be treated to give the anticipated physiological response.

The term “physiologically effective amount” is that amount delivered toa subject to give the desired palliative or curative effect.

The term “pharmaceutically acceptable carrier” means that the carriercan be taken into the lungs with no significant adverse toxicologicaleffects on the lungs.

The types of pharmaceutical excipients that are useful as carrierinclude stabilizers such as human serum albumin (HSA), bulking agentssuch as carbohydrates, amino acids and polypeptides; pH adjusters orbuffers; salts such as sodium chloride; and the like. These carriers maybe in a crystalline or amorphous form or may be a mixture of the two.

Bulking agents that are particularly valuable include compatiblecarbohydrates, polypeptides, amino acids or combinations thereof.Suitable carbohydrates include monosaccharides such as galactose,D-mannose, sorbose, and the like; disaccharides, such as lactose,trehalose, and the like; yclodextrins, such as2-hydroxypropyl-.beta.-cyclodextrin; and polysaccharides, such asraffinose, maltodextrins, dextrans, and the like; alditols, such asmannitol, xylitol, and the like. A preferred group of carbohydratesincludes lactose, threhalose, raffinose maltodextrins, and mannitol.Suitable polypeptides include aspartame. Amino acids include alanine andglycine, with glycine being preferred.

Suitable pH adjusters or buffers include organic salts prepared fromorganic acids and bases, such as sodium citrate, sodium ascorbate, andthe like; sodium citrate is preferred.

Dosage. An iRNA agent can be administered at a unit dose less than about75 mg per kg of bodyweight, or less than about 70, 60, 50, 40, 30, 20,10, 5, 2, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001, or 0.0005 mg per kg ofbodyweight, and less than 200 mmole of RNA agent (e.g., about 4.4×10¹⁶copies) per kg of bodyweight, or less than 1500, 750, 300, 150, 75, 15,7.5, 1.5, 0.75, 0.15, 0.075, 0.015, 0.0075, 0.0015, 0.00075, 0.00015nmole of RNA agent per kg of bodyweight. The unit dose, for example, canbe administered by injection (e.g., intravenous or intramuscular,intrathecally, or directly into an organ), an inhaled dose, or a topicalapplication.

Delivery of an iRNA agent directly to an organ (e.g., directly to theliver) can be at a dosage on the order of about 0.00001 mg to about 3 mgper organ, or preferably about 0.0001-0.001 mg per organ, about 0.03-3.0mg per organ, about 0.1-3.0 mg per eye or about 0.3-3.0 mg per organ.

The dosage can be an amount effective to treat or prevent a disease ordisorder.

In one embodiment, the unit dose is administered less frequently thanonce a day, e.g., less than every 2, 4, 8 or 30 days. In anotherembodiment, the unit dose is not administered with a frequency (e.g.,not a regular frequency). For example, the unit dose may be administereda single time.

In one embodiment, the effective dose is administered with othertraditional therapeutic modalities.

In one embodiment, a subject is administered an initial dose, and one ormore maintenance doses of an iRNA agent, e.g., a double-stranded iRNAagent, or siRNA agent, (e.g., a precursor, e.g., a larger iRNA agentwhich can be processed into an siRNA agent, or a DNA which encodes aniRNA agent, e.g., a double-stranded iRNA agent, or siRNA agent, orprecursor thereof). The maintenance dose or doses are generally lowerthan the initial dose, e.g., one-half less of the initial dose. Amaintenance regimen can include treating the subject with a dose ordoses ranging from 0.01 μg to 75 mg/kg of body weight per day, e.g., 70,60, 50, 40, 30, 20, 10, 5, 2, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001, or0.0005 mg per kg of bodyweight per day. The maintenance doses arepreferably administered no more than once every 5, 10, or 30 days.Further, the treatment regimen may last for a period of time which willvary depending upon the nature of the particular disease, its severityand the overall condition of the patient. In preferred embodiments thedosage may be delivered no more than once per day, e.g., no more thanonce per 24, 36, 48, or more hours, e.g., no more than once every 5 or 8days. Following treatment, the patient can be monitored for changes inhis condition and for alleviation of the symptoms of the disease state.The dosage of the compound may either be increased in the event thepatient does not respond significantly to current dosage levels, or thedose may be decreased if an alleviation of the symptoms of the diseasestate is observed, if the disease state has been ablated, or ifundesired side-effects are observed.

The effective dose can be administered in a single dose or in two ormore doses, as desired or considered appropriate under the specificcircumstances. If desired to facilitate repeated or frequent infusions,implantation of a delivery device, e.g., a pump, semi-permanent stent(e.g., intravenous, intraperitoneal, intracisternal or intracapsular),or reservoir may be advisable.

In one embodiment, the iRNA agent pharmaceutical composition includes aplurality of iRNA agent species. The iRNA agent species can havesequences that are non-overlapping and non-adjacent with respect to anaturally occurring target sequence. In another embodiment, theplurality of iRNA agent species is specific for different naturallyoccurring target genes. In another embodiment, the iRNA agents arespecific for different alleles.

Following successful treatment, it may be desirable to have the patientundergo maintenance therapy to prevent the recurrence of the diseasestate, wherein the compound of the invention is administered inmaintenance doses, ranging from 0.01 μg to 100 g per kg of body weight(see U.S. Pat. No. 6,107,094).

The concentration of the iRNA agent composition is an amount sufficientto be effective in treating or preventing a disorder or to regulate aphysiological condition in humans. The concentration or amount of iRNAagent administered will depend on the parameters determined for theagent and the method of administration, e.g. nasal, buccal, orpulmonary. For example, nasal formulations tend to require much lowerconcentrations of some ingredients in order to avoid irritation orburning of the nasal passages. It is sometimes desirable to dilute anoral formulation up to 10-100 times in order to provide a suitable nasalformulation.

Certain factors may influence the dosage required to effectively treat asubject, including but not limited to the severity of the disease ordisorder, previous treatments, the general health and/or age of thesubject, and other diseases present. Moreover, treatment of a subjectwith a therapeutically effective amount of an iRNA agent, e.g., adouble-stranded iRNA agent, or siRNA agent (e.g., a precursor, e.g., alarger iRNA agent which can be processed into an siRNA agent, or a DNAwhich encodes an iRNA agent, e.g., a double-stranded iRNA agent, orsiRNA agent, or precursor thereof) can include a single treatment or,preferably, can include a series of treatments. It will also beappreciated that the effective dosage of an iRNA agent such as an siRNAagent used for treatment may increase or decrease over the course of aparticular treatment. Changes in dosage may result and become apparentfrom the results of diagnostic assays as described herein. For example,the subject can be monitored after administering an iRNA agentcomposition. Based on information from the monitoring, an additionalamount of the iRNA agent composition can be administered.

Dosing is dependent on severity and responsiveness of the diseasecondition to be treated, with the course of treatment lasting fromseveral days to several months, or until a cure is effected or adiminution of disease state is achieved. Optimal dosing schedules can becalculated from measurements of drug accumulation in the body of thepatient. Persons of ordinary skill can easily determine optimum dosages,dosing methodologies and repetition rates. Optimum dosages may varydepending on the relative potency of individual compounds, and cangenerally be estimated based on EC50s found to be effective in in vitroand in vivo animal models. In some embodiments, the animal modelsinclude transgenic animals that express a human gene, e.g., a gene thatproduces a target RNA. The transgenic animal can be deficient for thecorresponding endogenous RNA. In another embodiment, the composition fortesting includes an iRNA agent that is complementary, at least in aninternal region, to a sequence that is conserved between the target RNAin the animal model and the target RNA in a human.

The invention is further illustrated by the following examples, whichshould not be construed as further limiting.

EXAMPLES Bio-Cleavable Oligonucleotide-Ligand Conjugates for TherapeuticApplications

Oligonucleotide Ligand Conjugate

NA is a chemically modified or unmodified oligonucleotide (or a nucleicacid) comprising of either RNA or DNA or chimeric RNA-DNA, DNA-RNA,RNA-DNA-RNA or DNA-RNA-DNA. L is ligand (Table 2), X is chemical linkagebetween the ligand and tether (see Table 3); Y is chemical linkagebetween tether and linker (see Table 3), and Z is chemical linkagebetween linker and oligonucleotide (see Table 3). Also see Table 3 fordefinition of tether.

TABLE 2 Definition of ligand L.

L = Cholesterol Thiocholesterol 5β-Cholanic Acid Cholic acid Lithocholicacid Biotin Vitamin E Naproxen Ibuprofen Amines (mono, di, tri,tetraalkyl or aryl) Folate Sugar (N-Acetylgalactosamine, galactosamine,galactose, Mannose) —(CH₂)_(n)NQ₁Q₂, where n = 0–40, Q₁, Q₂ = H, Me orEt; Q₁ = H, Q₂ = H, Me, Et or aryl —(CH₂)_(p)CH═CH(CH₂)_(q)NQ₁Q₂, wherep and/or q = 0–40, Q₁, Q₂ = H, Me or Et; Q₁ = H, Q₂ = H, Me, Et or arylwith E and/or Z configuration (CH₂)_(p)CH═(CH₂)_(q)NQ₁Q₂, where p and/orq = 0–40, Q₁, Q₂ = H, Me or Et; Q₁ = H, Q₂ = H, Me, Et or aryl(CH₂)_(p)CH═CH(CH₂)_(q)CH═CH(CH₂)_(r)NQ₁Q₂, where p, q and/or r = 0–40,Q₁, Q₂ = H, Me or Et; Q₁ = H, Q₂ = H, Me, Et or aryl with E and/or Zconfiguration —O(CH₂)_(m)(OCH₂CH₂)_(n)—OR, where m, n = 014 40 and R =H, Me, NQ₁Q₂, —C(O)NR′R″ —C(S)NR′R″ —NH(CH₂)_(m)(OCH₂CH₂)_(n)—OR, wherem, n = 0–40 and R = H, Me, NQ₁Q₂, —C(O)NR′R″ —C(S)NR′R″—O(CH₂)_(m)(NHCH₂CH₂)_(n)-R, where m, n = 0–40 and R = H, OH, Me, NQ₁Q₂,—C(O)NR′R″ —C(S)NR′R″ NH(CH₂)_(m)(NHCH₂CH₂)_(n)-R, where m, n = 0–40 andR = H, OH, Me, NQ₁Q₂, —C(O)NR′R″ —C(S)NR′R″ Dialkylglycerol (sn3, sn1,sn2 and racemic) with number of methylene varies from 0–40Diacylglycerol (sn3, sn1, sn2 and racemic) with number of methylenevaries from 0–40 Dialkylglycerol (sn3, sn1, sn2 and racemic) with numberof methylene varies from 0–40 and the alkyl chian contains one or moredouble bonds with E and/or Z isomers Diacylglycerol (sn3, sn1, sn2 andracemic) with number of methylene varies from 0–40 and the alkyl chiancontains one or more double bonds with E and/or Z isomers Lipids

Table 3. Definition of tether.

X = —NHC(O)— Y = —NHC(O)— Z = —NHC(O)— —C(O)NH— —C(O)NH— —C(O)NH——OC(O)NH— —OC(O)NH— —OC(O)NH— —NHC(O)O— —NHC(O)O— —NHC(O)O— —O— —O— —O——S— —S— —S— —SS— —SS— —SS— —S(O)— —S(O)— —S(O)— —S(O₂)— —S(O₂)— —S(O₂)——NHC(O)NH— —NHC(O)NH— —NHC(O)NH— —NHC(S)NH— —NHC(S)NH— —NHC(S)NH——C(O)O— —C(O)O— —C(O)O— —OC(O)— —OC(O)— —OC(O)— —NHC(S)— —NHC(S)——NHC(S)— —NHC(S)O— —NHC(S)O— —NHC(S)O— —C(S)NH— —C(S)NH— —C(S)NH——OC(S)NH— —OC(S)NH— —OC(S)NH— —NHC(S)O— —NHC(S)O— —NHC(S)O— —CH₂— —CH₂——CH₂— —CH₂CH═CH— —CH₂CH═CH— —CH₂CH═CH— —C(O)CH═CH— —C(O)CH═CH——C(O)CH═CH— —NH—CH₂CH═CH— —NH—CH₂CH═CH— —NH—CH₂CH═CH— —O—P(O)(OH)—O——O—P(O)(OH)—O— —O—P(O)(OH)—O— —O—P(S)(OH)—O— —O—P(S)(OH)—O——O—P(S)(OH)—O— —O—P(S)(SH)—O— —O—P(S)(SH)—O— —O—P(S)(SH)—O——S—P(O)(OH)—O— —S—P(O)(OH)—O— —S—P(O)(OH)—O— —O—P(O)(OH)—S——O—P(O)(OH)—S— —O—P(O)(OH)—S— —S—P(O)(OH)—S— —S—P(O)(OH)—S——S—P(O)(OH)—S— —O—P(S)(OH)—S— —O—P(S)(OH)—S— —O—P(S)(OH)—S——S—P(S)(OH)—O— —S—P(S)(OH)—O— —S—P(S)(OH)—O— —O—P(O)(R)—O— —O—P(O)(R)—O——O—P(O)(R)—O— —O—P(S)(R)—O— —O—P(S)(R)—O— —O—P(S)(R)—O— —S—P(O)(R)—O——S—P(O)(R)—O— —S—P(O)(R)—O— —S—P(S)(R)—O— —S—P(S)(R)—O— —S—P(S)(R)—O——S—P(O)(R)—S— —S—P(O)(R)—S— —S—P(O)(R)—S— —O—P(S)(R)—S— —O—P(S)(R)—S——O—P(S)(R)—S— R = Alkyl, fluroalkyl, aryl or aralkyl

Scheme 11: Synthesis of Thiocholesterol—Hydroxyprolinol Buildign BlocksContaining a Disulfide Linkage

Example 1 Synthesis of Compound 5 (Scheme 1)

Step 1, Compound 2: Compound 1 (20.37 g, 86.64 mmol, purchased fromAldrich) and 4-(dimethylamino)pyridine (DMAP, 1.40 g, 11.46 mmol) weredried over anhydrous P₂O₅ under vacuum overnight. After releasing vacuumunder argon, the mixture was then taken into anhydrous pyridine (150 mL)and to this DMTr-Cl (36.7 g, 108.3 mmol) was added at ambienttemperature. The reaction mixture was stirred at ambient temperatureovernight. After removing pyridine in vacuo, the product was extractedinto ethyl acetate (600 mL), washed with water and sodium bicarbonatesolution followed by standard workup. Compound 2 was purified by flashsilica gel column chromatography, eluent: 3-4% methanol indichloromethane, yield: 26.76 g (55.9%). ¹H NMR (400 MHz, [D₆]DMSO, 25°C.): δ 7.38-7.04 (m, 14H); 6.86-6.84 (d, 4H); 5.06 (bs, 1H, exchangeablewith D₂O); 4.97-4.89 (m, 2H); 4.33-4.28 (bm, 1H); 4.02-3.97 (bm, 1H);3.68 S, 6H); 3.47-3.38 (m, 2H); 3.21-3.02 (m, 2H); 2.03-1.79 (m, 2H).

Step 2, Compound 3: Compound 2 (12.1 g, 21.87 mmol) in anhydrouspyridine (60 mL) was stirred with TBDMS-—Cl (4.90 g, 32.51 mmol) in thepresence of imidazole (6.0 g, 88.13 mmol) at ambient temperatureovernight. Pyridine was removed form the reaction mixture in vacuo andthe product was extracted into ethyl acetate (200 mL), washed withsodium bicarbonate solution followed by standard workup. Compound 3 waspurified by flash silica gel column chromatography, eluent: 1-2%methanol in dichloromethane; yield: 14.40 g (95.9%) (Corey andVenkkateswarlu, J. Am. Chem. Soc., 1972, 94, 6190).

Step 3, Compound 5: Compound 3 and 10% palladium on carbon (wet, Degussatype, 10% by weight with respect to 3) are suspended in a 9:1 mixture ofethyl acetate-methanol and hydrogenated at 1 atm pressure to remove thebenzyl carbamate protection from compound 3 (Step 3a). After completedeprotection, the product is separated from the catalyst by filtration.Solvent is removed from the filtrate in vacuo and the product isco-evaporated with anhydrous dichloromethane and re-dissolved inanhydrous dichloromethane. To this one molar equivalent ofN,N′-disuccinimidyl carbonate (DSC) and anhydrous triethylamine (TEA)are added the reaction mixture is stirred for overnight (Takeda et al.,Tetrahedron Lett., 1983, 24, 4569). After overnight stirring cystaminedihydrochloride (purchased from Aldrich) and excess of anhydrous TEA areadded to the stirring solution, continued stirring to obtain compound 5(Step 3b).

Example 2 Synthesis of Compound 6 (Scheme 1)

Step 1, Compound 4: Adipic acid monomethyl ester is treated with onemolar equivalent of DCC, DMAP and N-hydroxysuccinimide in DMF for 30min. To this a molar equivalent of the compound obtained from Step 3a(Example 1) is added and stirred at ambient temperature for 8 h.Dicylcohexylurea formed during the course of the reaction is filteredoff and the product is extracted into ethyl acetate, washed with sodiumbicarbonate solution followed by standard workup. The methyl ester thusobtained is treated with LiOH in THF/water to obtain compound 4.

Step 2, Compound 6: Compound 4 is treated with DCC, DMAP andN-hydroxysuccinimide as described in step 1 above and subsequentlyreacted with cystamine dihydrochloride in the presence of TEA to obtaincompound 6.

Example 3 Synthesis of Compound 10 (Scheme 1)

Step 1, Compound 7: Compound 1 in pyridine in reacted with TBDMS—Cl (1mol eq.) in the presence of imidazole (2 mol eq.) to obtain compound 7.

Step 2, Compound 8: Compound 7 is reacted with DMTr-Cl in anhydrouspyridine in the presence of DMAP to obtain compound 7.

Step 3, Compound 10: The benzyl carbamate protection on compound 7 isremoved as described in Step 3a in Example 1. The product thus obtainedis reacted with cystamine dihydrochloride as described in Step 3b inExample 1 to obtain compound 10.

Example 4 Synthesis of Compound 11 (Scheme 1)

Compound 11 is prepared from compound 8 as described in Example 2.

Example 5 Synthesis of Compound 12 (Scheme 2)

Compound 5 is treated with DSC (1 mol eq.) in the presence of TEA indichloromethane and subsequently with cystamine dihydrochloride in thepresence of excess of TEA as described in step 3, Example 1 to obtaincompound 12.

Example 6 Synthesis of Compound 13 (Scheme 2)

Compound 10 is treated with DSC (1 mol eq.) in the presence of TEA indichloromethane and subsequently with cystamine dihydrochloride in thepresence of excess of TEA as described in step 3, Example 1 to obtaincompound 13.

Example 7 Syntheses of Compounds 14a-f (Scheme 3)

Step 1, Compound 14a: Naproxen (14, purchased from Aldrich) is stirredwith DCC (1 mol eq.), DMAP (0.1 mol eq.) and N-hydroxysuccinimide (2 moleq.) in dichlromethane for 30 min and to this compound 5 and TEA areadded to obtain compound 14a.

Step 2, Compound 14b-f are prepared from 14 as described in step 1 bysubsequent reaction of the activated carboxylate obtained by thereaction of DCC and N-hydroxysuccinimide in the presence of DMAP withcompound 6, 10, 11, 12 and 13 respectively.

Example 8 Syntheses of Compounds 15a-f (Scheme 3)

Step 1, Compound 15a: Compound 15 (purchased from Aldrich) is stirredwith DCC (1 mol eq.), DMAP (0.1 mol eq.) and N-hydroxysuccinimide (2 moleq.) in dichlromethane for 30 min and to this compound 5 and TEA areadded to obtain compound 15a.

Step 2, Compound 15b-f are prepared from 15 as described in step 1 bysubsequent reaction of the activated carboxylate obtained by thereaction of DCC and N-hydroxysuccinimide in the presence of DMAP withcompound 6, 10, 11, 12 and 13 respectively.

Example 9 Syntheses of Compounds 16a-f (Scheme 3)

Step 1, Compound 16a: Compound 16 (purchased from Aldrich) is stirredwith DCC (1 mol eq.), DMAP (0.1 mol eq.) and N-hydroxysuccinimide (2 moleq.) in dichlromethane for 30 min and to this compound 5 and TEA areadded to obtain compound 16a.

Step 2, Compound 16b-f are prepared from 16 as described in step 1 bysubsequent reaction of the activated carboxylate obtained by thereaction of DCC and N-hydroxysuccinimide in the presence of DMAP withcompound 6, 10, 11, 12 and 13 respectively.

Example 10 Syntheses of Compounds 17a-f (Scheme 3)

Step 1, Compound 17a: Compound 17 is prepared as reported in theliterature (Fang and Bergstrom, Nucleic Acids Res., 2003, 31, 708).Compound 17 is stirred with DCC (1 mol eq.), DMAP (0.1 mol eq.) andN-hydroxysuccinimide (2 mol eq.) in dichlromethane for 30 min and tothis compound 5 and TEA are added to obtain compound 17a.

Step 2, Compound 17b-f are prepared from 17 as described in step 1 bysubsequent reaction of the activated carboxylate obtained by thereaction of DCC and N-hydroxysuccinimide in the presence of DMAP withcompound 6, 10, 11, 12 and 13 respectively.

Example 11 Syntheses of Compounds 18a-f (Scheme 3)

Step 1, Compound 18a: Compound 18 is prepared as reported in theliterature (Rahal and Badache, Journal de la Societe Algerienne deChemie, 1994, 4, 75). Compound 18 is stirred with DCC (1 mol eq.), DMAP(0.1 mol eq.) and N-hydroxysuccinimide (2 mol eq.) in dichlromethane for30 min and to this compound 5 and TEA are added to obtain compound 18a.

Step 2, Compound 18b-f are prepared from 18 as described in step 1 bysubsequent reaction of the activated carboxylate obtained by thereaction of DCC and N-hydroxysuccinimide in the presence of DMAP withcompound 6, 10, 11, 12 and 13 respectively.

Example 12 Syntheses of Compounds 19a-f (Scheme 3)

Step 1, Compound 19a: Commercially available compound 19 is stirred withDCC (1 mol eq.), DMAP (0.1 mol eq.) and N-hydroxysuccinimide (2 mol eq.)in dichlromethane for 30 min and to this compound 5 and TEA are added toobtain compound 19a.

Step 2, Compound 19b-f are prepared from 19 as described in step 1 bysubsequent reaction of the activated carboxylate obtained by thereaction of DCC and N-hydroxysuccinimide in the presence of DMAP withcompound 6, 10, 11, 12 and 13 respectively.

Example 13 Syntheses of Compounds 20a-f (Scheme 3)

Step 1, Compound 20a: Compound 20 is prepared as reported in theliterature (De et al., 1998, 41, 4918). Compound 20 is stirred with DCC(1 mol eq.), DMAP (0.1 mol eq.) and N-hydroxysuccinimide (2 mol eq.) indichlromethane for 30 min and to this compound 5 and TEA are added toobtain compound 20a.

Step 2, Compound 20b-f are prepared from 20 as described in step 1 bysubsequent reaction of the activated carboxylate obtained by thereaction of DCC and N-hydroxysuccinimide in the presence of DMAP withcompound 6, 10, 11, 12 and 13 respectively.

Example 14 Syntheses of Compounds 21a-f (Scheme 3)

Step 1, Compound 21a: Commercially available 5β-cholanic acid 21 isstirred with DCC (1 mol eq.), DMAP (0.1 mol eq.) andN-hydroxysuccinimide (2 mol eq.) in dichlromethane for 30 min and tothis compound 5 and TEA are added to obtain compound 21a.

Step 2, Compound 21b-f are prepared from 21 as described in step 1 bysubsequent reaction of the activated carboxylate obtained by thereaction of DCC and N-hydroxysuccinimide in the presence of DMAP withcompound 6, 10, 11, 12 and 13 respectively.

Example 15 Syntheses of Compounds 22a-f (Scheme 3)

Step 1, Compound 22a: Compound 22 is stirred with DCC (1 mol eq.), DMAP(0.1 mol eq.) and N-hydroxysuccinimide (2 mol eq.) in dichlromethane for30 min and to this compound 5 and TEA are added to obtain compound 22a.

Step 2, Compound 22b-f are prepared from 22 as described in step 1 bysubsequent reaction of the activated carboxylate obtained by thereaction of DCC and N-hydroxysuccinimide in the presence of DMAP withcompound 6, 10, 11, 12 and 13 respectively.

Example 16 Syntheses of Compounds 22a-f (Scheme 3)

Step 1, Compound 23a: Compound 23 is obtained as reported in theliterature (Valentijn et al., Tetrahedron, 1997, 53, 759). Compound 23is stirred with DCC (1 mol eq.), DMAP (0.1 mol eq.) andN-hydroxysuccinimide (2 mol eq.) in dichlromethane for 30 min and tothis compound 5 and TEA are added to obtain compound 23a.

Step 2, Compound 23b-f are prepared from 23 as described in step 1 bysubsequent reaction of the activated carboxylate obtained by thereaction of DCC and N-hydroxysuccinimide in the presence of DMAP withcompound 6, 10, 11, 12 and 13 respectively.

Example 17 Syntheses of Compounds 24a-f (Scheme 3)

Step 1, Compound 24a: Compound 24 is obtained as reported in theliterature (Valentijn et al., Tetrahedron, 1997, 53, 759). Compound 24is stirred with DCC (1 mol eq.), DMAP (0.1 mol eq.) andN-hydroxysuccinimide (2 mol eq.) in dichlromethane for 30 min and tothis compound 5 and TEA are added to obtain compound 24a.

Step 2, Compound 24b-f are prepared from 24 as described in step 1 bysubsequent reaction of the activated carboxylate obtained by thereaction of DCC and N-hydroxysuccinimide in the presence of DMAP withcompound 6, 10, 11, 12 and 13 respectively.

Example 18 Syntheses of Compounds 25a-f (Scheme 3)

Step 1, Compound 25a: Compound 25 is stirred with DCC (1 mol eq.), DMAP(0.1 mol eq.) and N-hydroxysuccinimide (2 mol eq.) in dichlromethane for30 min and to this compound 5 and TEA are added to obtain compound 25a.

Step 2, Compound 25b-f are prepared from 25 as described in step 1 bysubsequent reaction of the activated carboxylate obtained by thereaction of DCC and N-hydroxysuccinimide in the presence of DMAP withcompound 6, 10, 11, 12 and 13 respectively.

Example 19 Syntheses of Compounds 26a-f (Scheme 4)

Step 1, Compound 26a: Compound 26 (Cholesterol) is stirred with DSC (1mol eq.) in dichloromethane in the presence of TEA overnight. Compound 5is added into the reaction mixture, after over night stirring andcontinued stirring to form compound 26a. Standard synthetic organicchemistry purification procedures yields compound 26a.

Step 2, Compound 26b-f: Compound 26 is initially reacted with DSC in thepresence of TEA and subsequently with compounds 6, 10, 11, 12 and 13 inthe presence of TEA to obtain their respective carbamates 26b-f.

Example 20 Syntheses of Compounds 27a-f (Scheme 4)

Step 1, Compound 27a: Commercially available compound 27 is stirred withDSC (1 mol eq.) in dichloromethane in the presence of TEA overnight.Compound 5 is added into the reaction mixture, after over night stirringand continued stirring to form compound 27a. Standard synthetic organicchemistry purification procedures yields compound 27a.

Step 2, Compound 27b-f: Compound 27 is initially reacted with DSC in thepresence of TEA and subsequently with compounds 6, 10, 11, 12 and 13 inthe presence of TEA to obtain their respective carbamates 27b-f.

Example 21 Syntheses of Compounds 28a-f (Scheme 4)

Step 1, Compound 28a: Commercially available compound 28 is stirred withDSC (1 mol eq.) in dichloromethane in the presence of TEA overnight.Compound 5 is added into the reaction mixture, after over night stirringand continued stirring to form compound 28a. Standard synthetic organicchemistry purification procedures yields compound 28a.

Step 2, Compound 28b-f: Compound 28 is initially reacted with DSC in thepresence of TEA and subsequently with compounds 6, 10, 11, 12 and 13 inthe presence of TEA to obtain their respective carbamates 28b-f.

Example 22 Syntheses of Compounds 29a-f (Scheme 4)

Step 1, Compound 29a: Compound 29 is stirred with DSC (1 mol eq.) indichloromethane in the presence of TEA overnight. Compound 5 is addedinto the reaction mixture, after over night stirring and continuedstirring to form compound 29a. Standard synthetic organic chemistrypurification procedures yields compound 29a.

Step 2, Compound 29b-f: Compound 29 is initially reacted with DSC in thepresence of TEA and subsequently with compounds 6, 10, 11, 12 and 13 inthe presence of TEA to obtain their respective carbamates 29b-f.

Example 23 Syntheses of Compounds 30a-f (Scheme 4)

Step 1, Compound 30a: Compound 30 is stirred with DSC (1 mol eq.) indichloromethane in the presence of TEA overnight. Compound 5 is addedinto the reaction mixture, after over night stirring and continuedstirring to form compound 30a. Standard synthetic organic chemistrypurification procedures yields compound 30a.

Step 2, Compound 30b-f: Compound 30 is initially reacted with DSC in thepresence of TEA and subsequently with compounds 6, 10, 11, 12 and 13 inthe presence of TEA to obtain their respective carbamates 30b-f.

Example 24 Syntheses of Compounds 31a-f (Scheme 4)

Step 1, Compound 31a: Compound 31 is stirred with DSC (1 mol eq.) indichloromethane in the presence of TEA overnight. Compound 5 is addedinto the reaction mixture, after over night stirring and continuedstirring to form compound 31a. Standard synthetic organic chemistrypurification procedures yields compound 31a.

Step 2, Compound 31b-f: Compound 31 is initially reacted with DSC in thepresence of TEA and subsequently with compounds 6, 10, 11, 12 and 13 inthe presence of TEA to obtain their respective carbamates 31b-f.

Example 25 Syntheses of Compounds 32a-f (Scheme 4)

Step 1, Compound 32a: Compound 32 is stirred with DSC (1 mol eq.) indichloromethane in the presence of TEA overnight. Compound 5 is addedinto the reaction mixture, after over night stirring and continuedstirring to form compound 32a. Standard synthetic organic chemistrypurification procedures yields compound 32a.

Step 2, Compound 32b-f: Compound 32 is initially reacted with DSC in thepresence of TEA and subsequently with compounds 6, 10, 11, 12 and 13 inthe presence of TEA to obtain their respective carbamates 32b-f.

Example 26 Syntheses of Compounds 33a-f (Scheme 4)

Step 1, Compound 33a: Compound 33 is obtained as reported in theliterature (Valentijn et al., Tetrahedron, 1997, 53, 759). Compound 33is stirred with DSC (1 mol eq.) in dichloromethane in the presence ofTEA overnight. Compound 5 is added into the reaction mixture, after overnight stirring and continued stirring to form compound 33a. Standardsynthetic organic chemistry purification procedures yields compound 33a.

Step 2, Compound 33b-f: Compound 33 is initially reacted with DSC in thepresence of TEA and subsequently with compounds 6, 10, 11, 12 and 13 inthe presence of TEA to obtain their respective carbamates 33b-f.

Example 27 Syntheses of Compounds 34a-f (Scheme 4)

Step 1, Compound 34a: Compound 34 is obtained as reported in theliterature (Valentijn et al., Tetrahedron, 1997, 53, 759). Compound 34is stirred with DSC (1 mol eq.) in dichloromethane in the presence ofTEA overnight. Compound 5 is added into the reaction mixture, after overnight stirring and continued stirring to form compound 34a. Standardsynthetic organic chemistry purification procedures yields compound 34a.

Step 2, Compound 34b-f: Compound 34 is initially reacted with DSC in thepresence of TEA and subsequently with compounds 6, 10, 11, 12 and 13 inthe presence of TEA to obtain their respective carbamates 34b-f.

Example 28 Syntheses of Compounds 35a-f (Scheme 4)

Step 1, Compound 35a: Compound 35 is obtained as reported in theliterature (Wijsman et al., Recueil des Travaux Chimiques des Pays-Bas,1996, 115, 397). Compound 35 is stirred with DSC (1 mol eq.) indichloromethane in the presence of TEA overnight. Compound 5 is addedinto the reaction mixture, after over night stirring and continuedstirring to form compound 35a. Standard synthetic organic chemistrypurification procedures yields compound 35a.

Step 2, Compound 35b-f: Compound 35 is initially reacted with DSC in thepresence of TEA and subsequently with compounds 6, 10, 11, 12 and 13 inthe presence of TEA to obtain their respective carbamates 35b-f.

Example 29 Synthesis of Compound 40 (Scheme 5)

Step 1, Compound 39: Commercially available dicarboxylic acid 38(purchased from Aldrich) in DMF is stirred with DCC (2 mol eq.), DMAP(0.1 mol eq.) and N-hydroxysuccinimide (3 mol eq.) to obtain the diester39.

Step 2, Compound 40: Compound 3 and 10% palladium on carbon (wet,Degussa type, 10% by weight with respect to 3) are suspended in a 9:1mixture of ethyl acetate-methanol and hydrogenated at 1 atm pressure toobtain compound 36 (step 2a). After complete deprotection, the productis separated from the catalyst by filtration. The free amine 36 (1 moleq.) thus obtained is stirred with compound 39 (1 mol eq.) in thepresence of TEA for 4 h and subsequently 1,6-diaminohexane and excessTEA are added into the stirring solution to obtain compound 40 (step2b).

Example 30 Synthesis of Compound 41 (Scheme 5)

Step 1, Compound 39: Compound 38 (purchased from Aldrich) in DMF isstirred with DCC (2 mol eq.), DMAP (0.1 mol eq.) andN-hydroxysuccinimide (3 mol eq.) to obtain the diester 39.

Step 2, Compound 41: Compound 8 and 10% palladium on carbon (wet,Degussa type, 10% by weight with respect to 8) are suspended in a 9:1mixture of ethyl acetate-methanol and hydrogenated at 1 atm pressure toobtain compound 37 (step 2a). After complete deprotection, the productis separated from the catalyst by filtration. The free amine 37 (1 moleq.) thus obtained is stirred with compound 39 (1 mol eq.) in thepresence of TEA for 4 h and subsequently 1,6-diaminohexane and excessTEA are added into the stirring solution to obtain compound 41 (step2b).

Example 31 Syntheses of Compounds 42a-b (Scheme 6)

Step 1, Compound 42a: Naproxen (14, purchased from Aldrich) is stirredwith DCC (1 mol eq.), DMAP (0.1 mol eq.) and N-hydroxysuccinimide (2 moleq.) in dichlromethane for 30 min and to this compound 40 and TEA areadded to obtain compound 42a.

Step 2, Compound 42b is prepared from 14 as described in step 1 bysubsequent reaction of the activated carboxylate obtained by thereaction of DCC and N-hydroxysuccinimide in the presence of DMAP withcompound 41.

Example 32 Syntheses of Compounds 43a-b (Scheme 6)

Step 1, Compound 43a: Compound 15 is stirred with DCC (1 mol eq.), DMAP(0.1 mol eq.) and N-hydroxysuccinimide (2 mol eq.) in dichlromethane for30 min and to this compound 40 and TEA are added to obtain compound 43a.

Step 2, Compound 43b is prepared from 15 as described in step 1 bysubsequent reaction of the activated carboxylate obtained by thereaction of DCC and N-hydroxysuccinimide in the presence of DMAP withcompound 41.

Example 33 Syntheses of Compounds 44a-b (Scheme 6)

Step 1, Compound 44a: Compound 16 is stirred with DCC (1 mol eq.), DMAP(0.1 mol eq.) and N-hydroxysuccinimide (2 mol eq.) in dichlromethane for30 min and to this compound 40 and TEA are added to obtain compound 44a.

Step 2, Compound 44b is prepared from 16 as described in step 1 bysubsequent reaction of the activated carboxylate obtained by thereaction of DCC and N-hydroxysuccinimide in the presence of DMAP withcompound 41.

Example 34 Syntheses of Compounds 45a-b (Scheme 6)

Step 1, Compound 45a: Compound 17 is stirred with DCC (1 mol eq.), DMAP(0.1 mol eq.) and N-hydroxysuccinimide (2 mol eq.) in dichlromethane for30 min and to this compound 40 and TEA are added to obtain compound 45a.

Step 2, Compound 45b is prepared from 17 as described in step 1 bysubsequent reaction of the activated carboxylate obtained by thereaction of DCC and N-hydroxysuccinimide in the presence of DMAP withcompound 41.

Example 35 Syntheses of Compounds 46a-b (Scheme 6)

Step 1, Compound 46a: Compound 18 is stirred with DCC (1 mol eq.), DMAP(0.1 mol eq.) and N-hydroxysuccinimide (2 mol eq.) in dichlromethane for30 min and to this compound 40 and TEA are added to obtain compound 46a.

Step 2, Compound 46b is prepared from 18 as described in step 1 bysubsequent reaction of the activated carboxylate obtained by thereaction of DCC and N-hydroxysuccinimide in the presence of DMAP withcompound 41.

Example 36 Syntheses of Compounds 47a-b (Scheme 6)

Step 1, Compound 47a: Compound 19 is stirred with DCC (1 mol eq.), DMAP(0.1 mol eq.) and N-hydroxysuccinimide (2 mol eq.) in dichlromethane for30 min and to this compound 40 and TEA are added to obtain compound 47a.

Step 2, Compound 47b is prepared from 19 as described in step 1 bysubsequent reaction of the activated carboxylate obtained by thereaction of DCC and N-hydroxysuccinimide in the presence of DMAP withcompound 41.

Example 37 Syntheses of Compounds 48a-b (Scheme 6)

Step 1, Compound 48a: Compound 20 is stirred with DCC (1 mol eq.), DMAP(0.1 mol eq.) and N-hydroxysuccinimide (2 mol eq.) in dichlromethane for30 min and to this compound 40 and TEA are added to obtain compound 48a.

Step 2, Compound 48b is prepared from 20 as described in step 1 bysubsequent reaction of the activated carboxylate obtained by thereaction of DCC and N-hydroxysuccinimide in the presence of DMAP withcompound 41.

Example 38 Syntheses of Compounds 49a-b (Scheme 6)

Step 1, Compound 49a: Compound 21 is stirred with DCC (1 mol eq.), DMAP(0.1 mol eq.) and N-hydroxysuccinimide (2 mol eq.) in dichlromethane for30 min and to this compound 40 and TEA are added to obtain compound 49a.

Step 2, Compound 49b is prepared from 21 as described in step 1 bysubsequent reaction of the activated carboxylate obtained by thereaction of DCC and N-hydroxysuccinimide in the presence of DMAP withcompound 41.

Example 39 Syntheses of Compounds 50a-b (Scheme 6)

Step 1, Compound 50a: Compound 22 is stirred with DCC (1 mol eq.), DMAP(0.1 mol eq.) and N-hydroxysuccinimide (2 mol eq.) in dichlromethane for30 min and to this compound 40 and TEA are added to obtain compound 50a.

Step 2, Compound 50b is prepared from 22 as described in step 1 bysubsequent reaction of the activated carboxylate obtained by thereaction of DCC and N-hydroxysuccinimide in the presence of DMAP withcompound 41.

Example 40 Syntheses of Compounds 51a-b (Scheme 6)

Step 1, Compound 51a: Compound 23 is stirred with DCC (1 mol eq.), DMAP(0.1 mol eq.) and N-hydroxysuccinimide (2 mol eq.) in dichlromethane for30 min and to this compound 40 and TEA are added to obtain compound 51a.

Step 2, Compound 51b is prepared from 23 as described in step 1 bysubsequent reaction of the activated carboxylate obtained by thereaction of DCC and N-hydroxysuccinimide in the presence of DMAP withcompound 41.

Example 41 Syntheses of Compounds 52a-b (Scheme 6)

Step 1, Compound 52a: Compound 24 is stirred with DCC (1 mol eq.), DMAP(0.1 mol eq.) and N-hydroxysuccinimide (2 mol eq.) in dichlromethane for30 min and to this compound 40 and TEA are added to obtain compound 52a.

Step 2, Compound 52b is prepared from 24 as described in step 1 bysubsequent reaction of the activated carboxylate obtained by thereaction of DCC and N-hydroxysuccinimide in the presence of DMAP withcompound 41.

Example 42 Syntheses of Compounds 53a-b (Scheme 6)

Step 1, Compound 53a: Compound 25 is stirred with DCC (1 mol eq.), DMAP(0.1 mol eq.) and N-hydroxysuccinimide (2 mol eq.) in dichlromethane for30 min and to this compound 40 and TEA are added to obtain compound 52a.

Step 2, Compound 53b is prepared from 25 as described in step 1 bysubsequent reaction of the activated carboxylate obtained by thereaction of DCC and N-hydroxysuccinimide in the presence of DMAP withcompound 41.

Example 43 Syntheses of Compounds 54a-b (Scheme 7)

Step 1, Compound 54a: Compound 26 is stirred with DSC (1 mol eq.) indichloromethane in the presence of TEA overnight. Compound 40 is addedinto the reaction mixture, after over night stirring and continuedstirring to form compound 54a. Standard synthetic organic chemistrypurification procedures yields compound 54a.

Step 2, Compound 54b: Compound 26 is initially reacted with DSC in thepresence of TEA and subsequently with compound 41 in the presence ofexcess TEA to obtain 54b.

Example 44 Syntheses of Compounds 55a-b (Scheme 7)

Step 1, Compound 55a: Compound 27 is stirred with DSC (1 mol eq.) indichloromethane in the presence of TEA overnight. Compound 40 is addedinto the reaction mixture, after over night stirring and continuedstirring to form compound 55a. Standard synthetic organic chemistrypurification procedures yields compound 55a.

Step 2, Compound 55b: Compound 27 is initially reacted with DSC in thepresence of TEA and subsequently with compound 41 in the presence ofexcess TEA to obtain 55b.

Example 45 Syntheses of Compounds 56a-b (Scheme 7)

Step 1, Compound 56a: Compound 28 is stirred with DSC (1 mol eq.) indichloromethane in the presence of TEA overnight. Compound 40 is addedinto the reaction mixture, after over night stirring and continuedstirring to form compound 56a. Standard synthetic organic chemistrypurification procedures yields compound 56a.

Step 2, Compound 56b: Compound 28 is initially reacted with DSC in thepresence of TEA and subsequently with compound 41 in the presence ofexcess TEA to obtain 56b.

Example 46 Syntheses of Compounds 57a-b (Scheme 7)

Step 1, Compound 57a: Compound 29 is stirred with DSC (1 mol eq.) indichloromethane in the presence of TEA overnight. Compound 40 is addedinto the reaction mixture, after over night stirring and continuedstirring to form compound 57a. Standard synthetic organic chemistrypurification procedures yields compound 57a.

Step 2, Compound 57b: Compound 29 is initially reacted with DSC in thepresence of TEA and subsequently with compound 41 in the presence ofexcess TEA to obtain 57b.

Example 47 Syntheses of Compounds 58a-b (Scheme 7)

Step 1, Compound 58a: Compound 30 is stirred with DSC (1 mol eq.) indichloromethane in the presence of TEA overnight. Compound 40 is addedinto the reaction mixture, after over night stirring and continuedstirring to form compound 58a. Standard synthetic organic chemistrypurification procedures yields compound 58a.

Step 2, Compound 58b: Compound 30 is initially reacted with DSC in thepresence of TEA and subsequently with compound 41 in the presence ofexcess TEA to obtain 58b.

Example 48 Syntheses of Compounds 58a-b (Scheme 7)

Step 1, Compound 58a: Compound 31 is stirred with DSC (1 mol eq.) indichloromethane in the presence of TEA overnight. Compound 40 is addedinto the reaction mixture, after over night stirring and continuedstirring to form compound 58a. Standard synthetic organic chemistrypurification procedures yields compound 58a.

Step 2, Compound 58b: Compound 31 is initially reacted with DSC in thepresence of TEA and subsequently with compound 41 in the presence ofexcess TEA to obtain 58b.

Example 49 Syntheses of Compounds 60a-b (Scheme 7)

Step 1, Compound 60a: Compound 32 is stirred with DSC (1 mol eq.) indichloromethane in the presence of TEA overnight. Compound 40 is addedinto the reaction mixture, after over night stirring and continuedstirring to form compound 60a. Standard synthetic organic chemistrypurification procedures yields compound 60a.

Step 2, Compound 60b: Compound. 32 is initially reacted with DSC in thepresence of TEA and subsequently with compound 41 in the presence ofexcess TEA to obtain 60b.

Example 50 Syntheses of Compounds 61a-b (Scheme 7)

Step 1, Compound 61a: Compound 33 is stirred with DSC (1 mol eq.) indichloromethane in the presence of TEA overnight. Compound 40 is addedinto the reaction mixture, after over night stirring and continuedstirring to form compound 61a. Standard synthetic organic chemistrypurification procedures yields compound 61a.

Step 2, Compound 61b: Compound 33 is initially reacted with DSC in thepresence of TEA and subsequently with compound 41 in the presence ofexcess TEA to obtain 61b.

Example 51 Syntheses of Compounds 62a-b (Scheme 7)

Step 1, Compound 62a: Compound 34 is stirred with DSC (1 mol eq.) indichloromethane in the presence of TEA overnight. Compound 40 is addedinto the reaction mixture, after over night stirring and continuedstirring to form compound 62a. Standard synthetic organic chemistrypurification procedures yields compound 62a.

Step 2, Compound 62b: Compound 34 is initially reacted with DSC in thepresence of TEA and subsequently with compound 41 in the presence ofexcess TEA to obtain 62b.

Example 52 Syntheses of Compounds 63a-b (Scheme 7)

Step 1, Compound 63a: Compound 35 is stirred with DSC (1 mol eq.) indichloromethane in the presence of TEA overnight. Compound 40 is addedinto the reaction mixture, after over night stirring and continuedstirring to form compound 63a. Standard synthetic organic chemistrypurification procedures yields compound 63a.

Step 2, Compound 63b: Compound 34 is initially reacted with DSC in thepresence of TEA and subsequently with compound 41 in the presence ofexcess TEA to obtain 63b.

Example 53 Syntheses of Compounds 64-151 (Scheme 8)

Step 1, Compound 64: Compound 14a is stirred with excesstetrabutylammonium fluoride in THF as reported in the literature toobtain compound 64 (Nakaba et al., Tetrahedron Lett., 1988, 29, 2219,2223).

Step 2, Compounds 65-151: The desired compounds 65-151 are obtained fromtheir corresponding precursor 15a-25a, 14c-25c, 14e-25e, 26a-35a,26c-35c, 26e-35e, 42a-53a and 54a-63a respectively as described in step1 for the synthesis of compound 64 from 14a.

Example 54 Syntheses of Compounds 152-239 (Scheme 8)

Step 1, Compound 152: Compound 64 is reacted with2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphine in the presence oftetrazolediisopropylammonium salt in anhydrous acetonitrile or in amixture of acetonitriel-dichloromethane as reported in the literature toobtain compound 152 (Rajeev et al., Org. Lett., 2003, 5, 3005).

Step 2, Compounds 153-239: The phosphoramidites 153-239 are preparedfrom their corresponding precursor 65-151 as described above for thepreparation compound 152 from 64.

Example 55 Syntheses of Compounds 240-327

Step 1, Compound 240: Treatment of compound 64 with succinic anhydridein the presence of DMAP as reported in the literature (Rajeev et al.,Org. Lett., 2003, 5, 3005) yields the corresponding succinate. Thesuccinate thus obtained is treated with 2,2′-dithiobis(5-nitropyridine)(purchased from Aldrich) and triphenylphosphine in the presence of DMAPfollowed by addition of long chain aminoalkyl CPG (from Millipore) asreported in the literature yields compound 240 (Kumar et al.,Nucleosides Nucleotides, 1996, 15, 879).

Step 2, Compounds 241-327: The soid supports 241-327 are obtained fromtheir corresponding precursor 65-151 as described above for thepreparation of compound 240 from compound 64.

Example 56 Syntheses of Compounds 328-415 (Scheme 8)

Step 1, Compound 328: Compound 14b is stirred with excesstetrabutylammonium fluoride in THF as reported in the literature toobtain compound 328 (Nakaba et al., Tetrahedron Lett., 1988, 29, 2219,2223).

Step 2, Compounds 329-415: The desired compounds 329-415 are obtainedfrom their corresponding precursor 15b-25b, 14d-25d, 14f-25f, 26b-35b,26d-35d, 26f-35f, 42b-53b and 54b-63b respectively as described in step1 for the synthesis of compound 328 from 14b.

Example 56 Syntheses of Compounds 416-503 (Scheme 8)

Step 1, Compound 416: Compound 328 is reacted with2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphine in the presence oftetrazolediisopropylammonium salt in anhydrous acetonitrile or in amixture of acetonitriel-dichloromethane as reported in the literature toobtain compound 416 (Rajeev et al., Org. Lett., 2003, 5, 3005).

Step 2, Compounds 417-503: The phosphoramidites 417-503 are preparedfrom their corresponding precursor 329-415 as described above for thepreparation compound 416 from 328.

Example 57 Syntheses of Compounds 504-691

Step 1, Compound 504: Treatment of compound 328 with succinic anhydridein the presence of DMAP as reported in the literature (Rajeev et al.,Org. Lett., 2003, 5, 3005) yields the corresponding succinate. Thesuccinate thus obtained is treated with 2,2′-dithiobis(5-nitropyridine)(purchased from Aldrich) and triphenylphosphine in the presence of DMAPfollowed by addition of long chain aminoalkyl CPG (from Millipore) asreported in the literature yields compound 504 (Kumar et al.,Nucleosides Nucleotides, 1996, 15, 879).

Step 2, Compounds 505-691: The soid supports 505-691 are obtained fromtheir corresponding precursor 329-415 as described above for thepreparation of compound 504 from compound 328.

Example 58 Synthesis of Compound 695 (Scheme 9)

Step 1, Compound 694: Compound 2 and 10% palladium on carbon (wet,Degussa type, 10% by weight with respect to 2) are suspended in a 9:1mixture of ethyl acetate-methanol and hydrogenated at 1 atm pressure toobtain compound 694.

Step 2, Compound 695: Compound 694 is stirred with commerciallyavailable 3-(2-pyridyldithio)propionic acid N-hydroyxysuccinimide ester(available from Bachem) in the presence of TEA in dichloromethane toobtain compound 695.

Example 59 Synthesis of Compound 696 (Scheme 9)

Phosphitylation of compound 695 as described in Example 56 yields thedesired phosphoramidite 696.

Example 60 Synthesis of Compound 697 (Scheme 9)

Compound 697 is obtained from compound 695 and long chain aminoalkyl CPG(from Millipore) as described in Example 57 for the preparation ofcompound 504 from compound 328.

Example 61 Synthesis of Compound 698 (Scheme 9)

Compound 695 is stirred with R—SH (any thiol or mercaptan, where R isany organic functional group or moiety) in the presence of TEA to obtaincompound 698.

Example 62 Synthesis of Compound 699 (Scheme 9)

Phosphitylation of compound 698 as described in Example 56 yields thedesired phosphoramidite 699.

Example 63 Synthesis of Compound 700 (Scheme 9)

Compound 700 is obtained from compound 698 and long chain aminoalkyl CPG(from Millipore) as described in Example 57 for the preparation ofcompound 504 from compound 328.

Example 64 Synthesis of Compound 702 (Scheme 10)

Compound 701 (7.7 g, 14.5 mmol) was dissolved in anhydrousdichloromethane (40 mL) and cooled to 0° C. To the solution were addedtriethylamine (3.0 g, 4.2 mL, 30 mmol) and3-(Pyridin-2-yldisulfanyl)-propionic succinate ester (SPDP) (4.5 g, 14.4mmol) successively. The reaction temperature was brought to ambienttemperature and stirred further for 16 h. The completion of the reactionwas ascertained by TLC (10% MeOH/CHCl₃, R_(f)=0.6). The reaction mixturewas diluted with dichloromethane and washed with saturated NaHCO₃, waterfollowed by brine. The organic layer was dried over sodium sulfate,filtered and concentrated under vacuum to afford the crude product.Compound 702 (10.58 g, 78%) was obtained as a white foamy solid aftercolumn chromatography over silica gel.

¹H NMR (400 MHz, DMSO-d₆): δ 8.45 (d, 1H), 7.9 (m, 1H), 7.8 (m, 1H),7.76 (m, 1H), 7.3 (m, 4H), 7.18 (m, 5H), 6.86 (m, 4H), 4.98 (d, —OH,1H), 4.38 (m, 1H), 4.1 (m, 1H) (s, 6H), 3.56 (m, 1H), 3.46 (m, 1H),3.21-3.34 (m, 3H), 3.14 (m, 1H), 3 (m, 2H), 2.48 (m, 2H), 2.2 (m, 2H),1.8-2.02 (m, 2H), 1.1-1.5 (4H).

¹³C NMR (100 MHz, DMSO-d₆): δ 171.32, 169.97, 159.36, 158.31, 158.18,149.80, 145.27, 138.08, 136.1, 135.9, 129.8, 128.0, 127.7, 121.4, 119.3,113.3, 85.338, 68.7, 55.3, 34.75, 34.28, 29.1, 26.3, 24.36.

Example 65 Synthesis of Compound 703 (Scheme 10)

Phosphitylation of compound 702 as described in Example 56 yields thedesired phosphoramidite 703.

Example 66 Synthesis of Compound 704 (Scheme 10)

Compound 704 is obtained from compound 702 and long chain aminoalkyl CPG(from Millipore) as described in Example 57 for the preparation ofcompound 504 from compound 328.

Example 67 Synthesis of Compound 705 (Scheme 10)

Compound 702 is stirred with R—SH (any thiol or mercaptan, where R isany organic functional group or moiety) in the presence of TEA to obtaincompound 705.

Example 68 Synthesis of Compound 706 (Scheme 10)

Phosphitylation of compound 705 as described in Example 56 yields thedesired phosphoramidite 706.

Example 69 Synthesis of Compound 700 (Scheme 9)

Compound 707 is obtained from compound 705 and long chain aminoalkyl CPG(from Millipore) as described in Example 57 for the preparation ofcompound 504 from compound 328.

Example 70 Synthesis of Compound 708 (Scheme 11)

Compound 702 (7.5 g, 10.28 mmol) was dissolved in anhydrousdichloromethane (75 mL) under argon and cooled to 0° C. To this solutionwere added diisopropylethyl amine (2.71 g, 3.66 mL, 21 mmol) followed bythiocholesterol (4.145 g, 10.28 mmol). The reaction mixture was broughtto ambient temperature and stirred further for 16 h. The completion ofthe reaction was ascertained by TLC (100% ethyl acetate, R_(f)=0.6). Thereaction mixture was concentrated under reduced pressure and the residuewas subjected to column chromatography on silica gel. Even though therewas good separation in hexane/ethyl acetate system, compoundprecipitates in that mixture. After eluting with 4 L of ethyl acetate,the column was eluted with 5% MeOH/dichloromethane (2 L) to obtaincompound 708 as white foamy solid (8 g, 76%).

¹H NMR (400 MHz, DMSO-d₆): δ 7.88 (m, 1H), 7.3 (m, 4H), 7.17 (m, 5H),6.84 (m, 4H), 5.3 (bs, 1H), 4.89 (d, —OH), 4.38 (m, 1H), 4.1 (m, 1H),3.72 (s, 6H), 3.56 (m, 1H), 3.32 (m, 1H), 3.14 (m, 1H), 3 (m, 3H), 2.84(m, 2H), 2.64 (m, 1H), 2.42 (m, 2H), 2.2 (m, 3H), 1.8-2.0 (m, 7H),0.8-1.54 (m, 35H), 0.62 (s, 3H).

¹³C NMR (100 MHz, DMSO-d₆): δ 170.8, 158.0, 157.9, 155.6, 145.0, 139.7,135.8, 135.7, 129.5, 127.7, 127.5, 121.7, 113.1, 113.0, 85.7, 85.1,72.7, 68.5, 63.3, 60.72, 56.1, 55.5, 55.28, 54.9, 49.4, 41.8, 36.5,35.2, 31.3, 30.35, 27.7, 27.3, 26.0, 24.1, 23.8, 23.2, 22.6, 22.3,21.11, 20.5, 19.43, 18.9, 18.5, 14.4, 11.6.

Example 71 Synthesis of Compound 709 (Scheme 11)

Compound 708 (5.7 g, 5.58 mmol) was coevaporated with anhydrous toluene(50 mL). To the residue N,N-tetraisopropylammonium tetrazolide (0.315 g,2.79 mmol) was added and the mixture was dried over P₂O₅ in a vacuumoven for overnight at 40° C. The reaction mixture was dissolved indichloromethane (20 mL) and2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphorodiamidite (2.48 g, 2.72mL, 8.25 mmol) was added. The reaction mixture was stirred at ambienttemperature for overnight. The completion of the reaction wasascertained by TLC (R_(f)=0.9 in ethyl acetate). The reaction mixturewas diluted with dichloromethane (50 mL) and washed with 5% NaHCO₃ (50mL) and brine (50 mL). The organic layer was dried over anhydrous Na₂SO₄filtered and concentrated under reduced pressure. The residue waspurified over silica gel (50:49:1, EtOAc:Hexane:triethlyamine) to afford709 as white foamy solid (6.1 g, 89%).

¹H NMR (400 MHz, C₆D₆): δ 7.62 (m, 2H), 7.45 (m, 5H), 7.24 (m, 2H), 7.1(m, 1H), 6.82 (m, 4H), 5.64 (m, 1H), 5.38 (m, 1H), 4.7 (m, 1H), 4.54 (m,2H), 3.78 (m, 2H), 3.5 (m, 3H), 3.36 (m, 9H), 3.22 (m, 4H), 3.06 (m,3H), 2.72 (m, 1H), 2.32-2.54 (m, 5H), 1.8-2.2 (m, 10H), 1.08-1.74 (m,28H), 1.3 (m, 6H), 0.94 (m, 12H), 0.67 (s, 3H).

³¹P NMR (161.82 MHz, C₆D₆): δ 146.05, 145.91, 145.66, 145.16

¹³C NMR (100 MHz, C₆D₆): δ 171.43, 171.25, 169.87, 159.25, 159.11,146.08, 141.59, 136.66, 136.6, 130.62, 130.54, 128.63, 127.53, 127.02,121.53, 117.73, 117.57, 113.66, 113.57, 86.59, 86.54, 64.36, 58.56,58.37, 58.30, 56.96, 56.51, 56.07, 54.86, 54.77, 50.57, 50.27, 43.48,43.35, 42.55, 40.13, 39.9, 39.75, 39.56, 38.70, 36.94, 36.64, 36.29,36.19, 35.90, 34.58, 32.24, 32.08, 29.48, 29.03, 28.98, 28.6, 28.38,26.54, 24.68, 24.61, 24.54, 23.6, 23.0, 22.74, 21.26, 20.03, 19.9,19.38, 19.01, 12.06.

Example 72 Synthesis of Compound 710 (Scheme 11)

Step 1, Synthesis of succinate of compound 708: Compound 708 (2.2 g,2.15 mmol) was mixed with succinic anhydride (0.323 g, 3.23 mmol) andDMAP (0.026 g, 0.215 mmol) and dried in a vacuum at 40° C. overnight.The mixture was dissolved in anhydrous dichloromethane (10 mL),triethylamine (0.708 g, 0.976 mL, 7 mmol) was added and the solution wasstirred at room temperature under argon atmosphere for 16 h. It was thendiluted with dichloromethane (50 mL) and washed with ice cold aqueouscitric acid (5% wt., 25 mL) and water (2×25 mL). The organic phase wasdried over anhydrous sodium sulfate and concentrated to dryness. Thecrude product was purified by column chromatography to afford thesuccinate derivative as white foamy solid (2.2 g, 92% yield; R_(f)=0.6 sin 10% MeOH/CHCl₃).

¹H NMR (400 MHz, DMSO-d₆): δ 12.22 (bs, 1H), 7.84 (m, 1H), 7.25 (m, 4H),7.2 (m, 5H), 6.86 (m, 4H), 5.36 (m, 2H), 4.18 (bs, 1H), 3.72 (s, 6H),3.4-3.6 (m, 2H), 3.2 (m, 1H), 3.0 (m, 4H), 2.84 (m, 2H), 2.64 (m, 2H),2.4-2.52 (m, 12H), 2.2 (m, 6H), 1.9 (m, 8H), 0.8-1.52 (m, 28H), 0.65 (s,3H).

¹³C NMR (100 MHz, DMSO-d₆): δ 173.35, 171.94, 170.63, 169.64, 157.99,144.96, 141.02, 135.72, 129.61, 127.81, 127.55, 113.12, 56.15, 54.99,52.28, 49.58, 49.06, 41.82, 36.17, 34.97, 33.41, 33.09, 31.32, 27.39,23.16, 22.68, 22.39, 20.56, 18.95, 18.54, 11.66, 6.02, 5.0

Step 2, Synthesis of compound 710 from the succinate: The succinate (2.1g, 1.9 mmol) thus obtained was dissolved in dichloroethane (8 mL). Tothat solution DMAP (0.228 g, 1.9 mmol) was added.2,2′-Dithio-bis(5-nitropyridine) (0.58 g, 1.9 mmol) inacetonitrile/dichloroethane (3:1, 8 mL) was added successively. To theresulting solution triphenylphosphine (0.49 g, 1.9 mmol) in acetonitrile(4 ml) was added. The reaction mixture turned bright orange in color.The solution was agitated briefly using wrist-action shaker (5 mins).Long chain alkyl amine-CPG (LCAA-CPG) (12 g, 1860 μmoles, 155 μm/g) wasadded. The suspension was agitated for 4 h. The CPG was filtered througha sintered funnel and washed with acetonitrile, dichloromethane andether successively. Unreacted amino groups were masked using aceticanhydride/pyridine. The loading capacity of the CPG 710 was measured bytaking UV measurement. (57 μM/g).

Example 73 Oligonucleotide Synthesis, Purification and Analysis

Synthesis:

The RNA molecules were synthesized on a 394 ABI machine using thestandard 93 step cycle written by the manufacturer with modifications toa few wait steps as described below. The solid support was available inhouse and the monomers were RNA phosphoramidites with fast protectinggroups (5′-O-dimethoxytritylN6-phenoxyacetyl-2′-O-t-butyldimethylsilyladenosine-3′-O—N,N′-diisopropyl-cyanoethylphosphoramidite,5′-O-dimethoxytrityl-N-4-acetyl-2′-O-t-butyldimethylsilylcytidine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite,5′-O-dimethoxytrityl-N-2-p-isopropylphenoxyacetyl-2′-O-t-butyldimethylsilylguanosine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite,and5′-O-dimethoxytrityl-2′-O-t-butyldimethylsilyluridine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramiditefrom Pierce Nucleic Acids Technologies. All 2′-O-Me amidites werereceived from Glen Research. All amidites were used at a concentrationof 0.15M in acetonitrile (CH₃CN) and a coupling time of 12-15 min. Theactivator was 5-(ethylthio)-1H-tetrazole (0.25M), for the PO-oxidationIodine/Water/Pyridine was used and for PS-oxidation, 2% Beaucage reagent(Iyer et al., J. Am. Chem. Soc., 1990, 112, 1253) in anhydrousacetonitrile was used. The sulphurization time was about 6 min.

Deprotection-I (Nucleobase Deprotection)

After completion of synthesis the support was transferred to a screw capvial (VWR Cat # 20170-229) or screw caps RNase free microfuge tube. Theoligonucleotide was cleaved from the support with simultaneousdeprotection of base and phosphate groups with 1.0 mL of a mixture ofethanolic ammonia [ammonia: ethanol (3:1)] for 15 h at 55° C. The vialwas cooled briefly on ice and then the ethanolic ammonia mixture wastransferred to a new microfuge tube. The CPG was washed with 2×0.1 mLportions of RNase free deionised water. Combined washings, cooled over adry ice bath for 10 min and subsequently dried in speed vac.

Deprotection-II (Removal of 2′ TBDMS Group)

The white residue obtained was resuspended in 400 μl of triethylamine,triethylamine trihydrofluoride (TEA.3HF) and NMP (4:3:7) and heated at50° C. for overnight to remove the tert-butyldimethylsilyl (TBDMS)groups at the 2′position (Wincott et al., Nucleic Acids Res., 1995, 23,2677). The reaction was then quenched with 400 μl ofisopropoxytrimethylsilane (iPrOMe₃Si, purchased from Aldrich) andfurther incubated on the heating block leaving the caps open for 10 min;(This causes the volatile isopropxytrimethylsilylfluoride adduct tovaporize). The residual quenching reagent was removed by drying in aspeed vac. Added 1.5 ml of 3% triethylamine in diethyl ether andpelleted by centrifuging. The supernatant was pipetted out withoutdisturbing the pellet and the pellet was dried in speed vac. The crudeRNA was obtained as a white fluffy material in the microfuge tube.

Quantitation of Crude Oligomer or Raw Analysis

Samples were dissolved in RNase free deionied water (1.0 mL) andquantitated as follows: Blanking was first performed with water alone (1mL) 20 μL of sample and 980 μL of water were mixed well in a microfugetube, transferred to cuvette and absorbance reading obtained at 260 nm.The crude material is dried down and stored at −20° C.

5. Purification of Oligomers:

PAGE Purification

PAGE purification of oligomers synthesized was performed as reported bySambrook et al. (Molecular Cloning: a Laboratory Manual, Second Edition,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).The 12% denaturing gel was prepared for purification of unmodified andmodified oligoribonucleotides. Took 120 mL Concentrate+105 mLDiluents+25 mL Buffer (National Diagnostics) then added 50 μL TEMED and1.5 mL 10% APS. Pour the gel and leave it for ½ h to polymerize.Suspended the RNA in 20 μL water and 80 μL formamide. Load the geltracking dye on left lane followed by the sample slowly on to the gel.Run the gel on 1×TBE buffer at 36 W for 4-6 h. Once run is completed,Transfer the gel on to preparative TLC plates and see under UV light.Cut the bands. Soak and crushed in Water. Leave in shaker for overnight.Remove the eluent, Dry in speed vac.

Desalting of Purified Oligomer

The purified dry oligomer was then desalted using Sephadex G-25 M(Amersham Biosciences). The cartridge was conditioned with 10 mL ofRNase free deionised water thrice. Finally the purified oligomer wasdissolved in 2.5 mL RNasefree water and passed through the cartridgewith very slow drop wise elution. The salt free oligomer was eluted with3.5 mL of RNase free water directly into a screw cap vial. Allolgonucleotides were finally analyzed by LC-MS and capillary gelelectrophoresis.

TABLE 4 List of ligand oligonucleotides (sense and antisense strand).Cal Mass Found Mass Sequence ID Sequence mu mu CGE (%) 711 5′ CUU ACGCUG AGU ACU UCG A dTdT 3′ 6606.00 6606.45 99.25 712 5′ UCG AAG UAC UCAGCG UAA G dT dT 3′ 6696.32 6693.0 89.0 713 5′ CUU ACG CUG AGU ACU UCG AdTdT L₁3′ 7312.96 7311.39 88.0 714 5′ UCG AAG UAC UCA GCG UAA G dT dT L₁3′ 7399.00 7399.06 92.00 715 5′ L₁ CUU ACG CUG AGU ACU UCG A dT dT 3′7311.88 7311.6 88.0 716 5′ L₁ UCG AAG UAC UCA GCG UAA G dT dT 3′ 7397.987396.2 95.2 717 5′ CUU ACG CUG AGU ACU UCG A dTdT L₂3′ 7387.39 7386.696.90 718 5′ UCG AAG UAC UCA GCG UAA G dT dT L₂ 3′ 7473.49 7474.0 92.00719 5′ L₂ CUU ACG CUG AGU ACU UCG A dT dT 3′ 7387.39 7787.53 90.00 7205′ L₂ UCG AAG UAC UCA GCG UAA G dT dT 3′ 7473.49 7473.54 96.34 L₁ =Cholesterol 6-aminohexanoic acid with trans-4-hydroxy-L-prolinol linkerL₂ = Thiocholesterol with trans-4-hydroxy-L-prolinol linker

Example 74 In Vitro Cell Culture Activities of Aromatic LigandConjugated siRNA Duplex

Dual Luciferase Gene Silencing Assays

In vitro activity of siRNAs was determined using a high-throughput96-well plate format luciferase silencing assay. Assays were performedin one of two possible formats. In the first format, HeLa SS6 cells werefirst transiently transfected with plasmids encoding firefly (target)and renilla (control) luciferase. DNA transfections were performed usingLipofectamine 2000 (Invitrogen) and the plasmids gWiz-Luc (Aldevron,Fargo, N. Dak.) (200 ng/well) and pRL-CMV (Promega, Madison Wis.) (200ng/well). After 2 h, the plasmid transfection medium was removed, andthe firefly luciferase targeting siRNAs were added to the cells atvarious concentrations. In the second format, HeLa Dual-luc cells(stably expressing both firefly and renilla luciferase) were directlytransfected with firefly luciferase targeting siRNAs. SiRNAtransfections were performed using either TransIT-TKO (Mirus, Madison,Wis.) or Lipofectamine 2000 according to manufacturer protocols. After24 h, cells were analyzed for both firefly and renilla luciferaseexpression using a plate luminometer (VICTOR², PerkinElmer, Boston,Mass.) and the Dual-Glo Luciferase Assay kit (Promega). Firefly/renillaluciferase expression ratios were used to determine percent genesilencing relative to mock-treated (no siRNA) controls.

TABLE 5 RNAi silencing by siRNA in stable Hela Dual Luc Cell Line:IC₅₀'s (nM) of duplexes tested with transfection agent. S Strand ASStrand 711 713 715 717 719 712 0.09 0.1 0.04 0.04 0.05 714 1.00 24716 >30.0 >30.00 718 0.08 0.20 2.0 720 0.04 2.0 0.13

TABLE 6 RNAi silencing by siRNA in transiently transfectd Hela Dual LucCell Line: IC₅₀'s (nM) of duplexes tested without transfection agent. SStrand AS Strand 711 713 715 717 719 712 >2000 30 20 <30.0 20.0 714260 >2000 716 >1500 >1500 718 <30.0 80.0 200 720 20. 200 40.0

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are within the scope of the followingclaims.

1. An iRNA agent comprising a first strand and optionally a secondstrand, wherein at least one subunit having a formula (I) isincorporated into at least one of the strands:

wherein: X is N(CO)R⁷, or NR⁷; Each of R¹, R³, and R⁹ is, independently,H, OH, OR^(a), OR^(b), CH₂OR^(a), or CH₂OR^(b), provided that at leastone of R¹, R³, and R⁹ is OH, OR^(a), or OR^(b) and that at least one ofR¹, R³, and R⁹ is CH₂OR^(a), or CH₂OR^(b); R⁷ is T-R^(d), in which T isa tether comprising a hydrocarbon chain and one or more linking groups,wherein at least one of the linking groups is cleavable; and R^(d) is asteroid radical, wherein the tether selected from the group consistingof

each of A and C is, independently, O or S; and B is OH, O⁻, or


2. The agent of claim 1, wherein at least one of the linking groups iscleaved at least about 100 times faster under intracellular conditionsthan under extracellular conditions.
 3. The agent of claim 1, wherein atleast one of the linking groups is cleaved at least about 50 timesfaster under intracellular conditions than under extracellularconditions.
 4. The agent of claim 1, wherein at least one of the linkinggroups is cleaved at least about 10 times faster under intracellularconditions than under extracellular conditions.
 5. The agent of claim 1,wherein R¹ is CH₂OR^(b) and R⁹ is OR^(a) or OH.
 6. The agent of claim 5,wherein R¹ and R⁹ are cis.
 7. The agent of claim 5, wherein R¹ and R⁹are trans.
 8. The agent of claim 1, wherein R¹ is OR^(a) and R⁹ isCH₂OR^(b).
 9. The agent of claim 8, wherein R¹ and R⁹ are cis.
 10. Theagent of claim 8, wherein R¹ and R⁹ are trans.
 11. The agent of claim 1,wherein R¹ is CH₂OR^(b) and R⁹ is OR^(b).
 12. The agent of claim 1,wherein R¹ is CH₂OR^(a) and R⁹ is OR^(b).
 13. The agent of claim 1,wherein R¹ is OR^(b) and R⁹ is CH₂OR^(b).
 14. The agent of claim 1,wherein R¹ is OR^(b) and R⁹ is CH₂OR^(a).
 15. The agent of claim 1,wherein the R^(d) steroid radical is a cholesterol radical.
 16. Theagent of claim 1, wherein the iRNA agent is a single-stranded iRNAagent.
 17. The agent of claim 16, wherein the single-stranded iRNA agentis an siRNA agent.
 18. The agent of claim 17, wherein R^(d) is acholesterol radical.
 19. The agent of claim 16, wherein the iRNA agentis less than 30 nucleotides in length.
 20. The agent of claim 19,wherein the iRNA agent is 21-23 nucleotides in length.
 21. The agent ofclaim 1, wherein the iRNA agent is a double-stranded iRNA agentcomprising a first strand and a second strand.
 22. The agent of claim21, wherein the double-stranded iRNA agent is an siRNA agent.
 23. Theagent of claim 22, wherein R^(d) is a cholesterol radical.
 24. The agentof claim 21, wherein the iRNA agent is less than 30 nucleotides inlength.
 25. The agent of claim 24, wherein the iRNA agent is 21-23nucleotides in length.
 26. The agent of claim 21, wherein the at leastone subunit of formula (I) is incorporated into both strands.
 27. Theagent of claim 1, wherein one of R³ or R⁹ is OH.
 28. The agent of claim1, wherein X is N(CO)R⁷.
 29. The agent of claim 1, wherein X is NR⁷. 30.The agent of claim 1, wherein R^(d) is uvaol.
 31. The agent of claim 1,wherein R^(d) is hecigenin.
 32. The agent of claim 1, wherein R^(d) isdiosgenin.
 33. The agent of claim 1, wherein R^(d) is cholic acid. 34.The agent of claim 1, wherein R^(d) is bile acid.
 35. The agent of claim1, wherein R^(d) is dihydrotestosterone.
 36. The agent of claim 1,wherein R^(d) is a cholesteryl residue.
 37. The agent of claim 1,wherein R^(d) is O3-(oleoyl)lithocholic acid radical.
 38. The agent ofclaim 1, wherein R^(d) is an O3-(oleoyl) cholenic acid radical.
 39. Theagent of claim 1, wherein A is O in each occurrence.
 40. The agent ofclaim 1, wherein A is S in at least one occurrence.
 41. The agent ofclaim 5, wherein R¹ is CH₂OR^(b) and R⁹ is OH.